Monoclonal antibodies specific for fibroblast growth factor receptor 4 (fgfr4) and methods of their use

ABSTRACT

Monoclonal antibodies selected from immunized mice, immunized rabbits and a human scFv library that specifically bind fibroblast growth factor receptor 4 (FGFR4) are described. Chimeric antigen receptors, antibody-drug conjugates, immunoconjugates, bispecific antibodies and immunoliposomes comprising the disclosed FGFR4-specific antibodies are also described. The antibody compositions can be used to diagnose or treat a FGFR4-positive cancer, such as rhabdomyosarcoma, lung cancer, liver cancer, breast cancer, pancreatic cancer or prostate cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/761,398, filed Mar. 19, 2018, which is the U.S. National Stage ofInternational Application No. PCT/US2016/052496, filed Sep. 19, 2016,published in English under PCT Article 21(2), which claims the benefitof U.S. Application No. 62/221,045, filed Sep. 20, 2015. Theabove-listed applications are herein incorporated by reference in theirentirety.

FIELD

This disclosure concerns monoclonal antibodies that specifically bindfibroblast growth factor receptor 4 (FGFR4) and conjugates thereof. Thisdisclosure further concerns use of the FGFR4-specific monoclonalantibodies and conjugates for the treatment of FGFR4-positive cancer.

BACKGROUND

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma inchildhood and adolescence, arising from skeletal myoblasts. There aretwo major subtypes of RMS—alveolar RMS (ARMS) and embryonal RMS (ERMS).With current treatment methods, relapse-free survival rates haveimproved to 70%-80%. However, the five-year survival rate for patientswith metastatic disease remains only 30%. The main drivers of tumorprogression and metastatic regulation are still unknown. It isspeculated, like in most cancers, there could be many factors that areinvolved in metastasis. One such factor known to play a role as ametastatic regulator is fibroblast growth factor receptor 4 (FGFR4).FGFR4, a member of the FGFR gene family, is a receptor tyrosine kinasethat is highly expressed in RMS.

Previous studies have associated activating mutations in FGFR4 to RMSmetastasis. FGFR4 is overexpressed in both subtypes of RMS, and in ARMS,the PAX3/7-FOX01 fusion gene directly induces FGFR4 expression. HighFGFR4 expression in RMS tumors is associated with advanced-stage cancer,an aggressive phenotype and poor survival. These findings suggest thatFGFR4 can be further exploited as a potential therapeutic target in RMS.Recent reports have also shown overexpression of FGFR4 in several otherhuman cancers including liver, lung, pancreas, ovary, prostate andbladder cancer.

SUMMARY

Disclosed herein are monoclonal antibodies, or antigen-binding fragmentsthereof, that specifically bind fibroblast growth factor receptor 4(FGFR4). The antibodies were selected from mice and rabbits immunizedwith the extracellular domain of human FGFR4 (hFGFR4-ECD), and from ahuman scFv library. Chimeric antigen receptors, antibody-drugconjugates, immunoconjugates, bispecific antibodies, immunoliposomes andcompositions comprising the FGFR4-specific antibodies are also disclosedherein. The monoclonal antibodies and antibody compositions can be usedto diagnose or treat a FGFR4-positive cancer, such as rhabdomyosarcoma,lung cancer, liver cancer, breast cancer, pancreatic cancer or prostatecancer.

Provided herein are monoclonal antibodies, or antigen-binding fragmentsthereof, that specifically bind FGFR4. In some embodiments, themonoclonal antibodies or antigen-binding fragments comprise the VHdomain and VL domain complementarity determining region (CDR) sequencesof a mouse antibody selected from BT53, 3A11 and 1G5, a rabbit antibodyselected from 29.2 and 57.1, or a human antibody selected from M408,M409, M410, M412, M414, M415, M417, M418, M422 and M424, as disclosedherein. Also provided herein are antibody drug conjugates (ADCs),chimeric antigen receptors (CARs), immunoconjugates, bispecificantibodies, immunoliposomes and compositions that include theFGFR4-specific monoclonal antibodies and antigen-binding fragments.Isolated cells expressing a CAR are also provided. Further providedherein are nucleic acid molecules and vectors encoding theFGFR4-specific monoclonal antibodies, antigen-binding fragments, CARs,immunoconjugates and bispecific antibodies disclosed herein.

Also provided herein is a method of inhibiting tumor growth ormetastasis of a FGFR4-positive cancer by selecting a subject with aFGFR4-positive cancer and administering to the subject a therapeuticallyeffective amount of a monoclonal antibody, antigen-binding fragment,ADC, CAR, isolated CAR-expressing cell, immunoconjugate, bispecificantibody, immunoliposome or composition disclosed herein. Furtherprovided is a method of treating a FGFR4-positive cancer in a subject byselecting a subject with a FGFR4-positive cancer and administering tothe subject a therapeutically effective amount of a monoclonal antibody,antigen-binding fragment, ADC, CAR, isolated CAR-expressing cell,immunoconjugate, bispecific antibody, immunoliposome or compositiondisclosed herein. In some embodiments, the FGFR-positive cancer is arhabdomyosarcoma (RMS), such as alveolar RMS or embryonal RMS, lungcancer, liver cancer, breast cancer, pancreatic cancer or prostatecancer.

A method of detecting expression of FGFR4 in a sample (such as a biopsysample) is also provided herein. In some embodiments, the methodincludes contacting the sample with a FGFR4-specific monoclonal antibodyor antigen-binding fragment disclosed herein and detecting binding ofthe antibody or antigen-binding fragment to the sample. In someexamples, the sample is obtained from a subject suspected of having aFGFR4-positive cancer.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of flow cytometry plots showing the specificity ofanti-FGFR4 mAbs binding to cell surface FGFR4. The murine RMS772 cellline was transfected with a plasmid containing the puromycin resistancegene alone (RMS772-puro) or a plasmid containing the puromycinresistance gene and the gene encoding full-length human wild type FGFR4(RMS772-FGFR4). Cells grown in selection medium were stained with 1μg/mL of anti-hFGFR4 monoclonal antibody from rabbit (29.2) or mouse(BT53, 3A11), and subsequently stained with fluorochrome-conjugatedsecondary antibody. Flow cytometry was performed using FACSCalibur. Allthree monoclonal antibodies exhibited significant binding to theFGFR4-transfected cells (non-filled histograms), but not to the vectorcontrol cells (filled histograms). Normal rabbit IgG and mouse IgG wereused as isotype controls.

FIGS. 2A-2B show that cell surface FGFR4 facilitates rapidinternalization of bound mAbs. RMS cell lines were incubated withsaturating amounts of mAb 29.2 or BT53 at 40° C. After washing, thecells were maintained at 40° C. (1), or further incubated at 37° C. forthe indicated time in the presence of 10 μM phenylarsine oxide (PAO)(2), or its diluent DMSO or medium only (3). Subsequently all cells werestained with fluorochrome-conjugated appropriate secondary antibody.FIG. 2A shows internalization that occurred at 2 hours in the indicatedcell lines. FIG. 2B indicates the percent monoclonal antibody retainedon the surface (MFI without incubation at 37° C. was set as 100%) duringthe time course of the experiment.

FIG. 3 is a graph showing FGFR4 protein expression in RMS cell lines issignificantly higher than in normal tissues. Whole cell lysates ofnormal tissues and RMS cell lines were normalized to total proteinconcentration of 1 mg/mL and tested on Meso Scale Discovery (MSD) assay(a noncompetitive sandwich assay). A standard curve was obtained usinghuman FGFR4 extracellular domain (ECD) protein. Samples were measuredbased on electrochemiluminescence signal.

FIG. 4 is a graph showing BT53 in conjunction with secondaryantibody-drug conjugate can mediate cytotoxicity in RMS cell lines. RMScell line (RH30) was incubated with differing amounts of mIgG or BT53mAb at the indicated concentrations. Subsequently, secondary ADC(anti-mouse-Fc-drug) was added at 6.6 nM. Dose-dependent cytotoxicactivity was observed following the addition of secondary ADC. Among thetwo drugs tested, duocarmycin DM (DMDM) showed more potent activity thanmonomethyl auristatin F (MMAF).

FIG. 5 is a pair of flow cytometry plots showing CAR expression intransduced T cells. Shown are expression of the FGFR4 CARs 29.2 L (left)and 57.1 L (right).

FIG. 6 is a series of graphs showing the results of cytotoxicity assaysusing T cells expressing the FGFR4 CARs 29.2 L (left) and 57.1 L.Percent lysis of rhabdomyosarcoma cells (RH41), osteosarcoma cells(143B) and myelogenous leukemia cells (K562) is shown.

FIG. 7 is a pair of graphs showing cytotoxicity mediated by anti-FGFR4monoclonal antibodies BT53 and 3A11 conjugated to secondaryantibody-drug conjugates in the RH30 cell line.

FIG. 8 is a pair of graphs showing cytotoxicity mediated by anti-FGFR4monoclonal antibody BT53 conjugated to secondary antibody-drugconjugates (ADC-MMAF—top; ADC-DMDM—bottom) in RMS-559 cells.

FIG. 9 is a pair of graphs showing specific cytotoxicity of anti-FGFR4monoclonal antibodies BT53 (top) and 3A11 (bottom) conjugated tosecondary antibody-drug conjugates. The FGFR4-specific secondaryantibody-drug conjugates induced killing of FGFR4-positiverhabdomyosarcoma cells (RH30), but not FGFR4-negative human skeletalmuscle cells (SKMC) cells.

FIGS. 10A-10B are graphs showing growth of FGFR-positive RH30 cells(FIG. 10A) and FGFR4-negative SKMC cells (FIG. 10B) in the presence ofthe BT53 monoclonal antibody and BT53 secondary ADC.

FIGS. 11A-11B are graphs showing cytotoxicity of T cells expressingFGFR4 CARs M410 long, M412 long and M412 short. A CD22 CAR was used as acontrol. Target RH30 (FGRR4+/CD22-) and Raji (FGFR4-/CD22+) cells weretransduced with luciferase and the CELLTITER-GLO™ assay was used tomeasure the number of viable cells. Percent specific lysis induced byeach CAR is shown. FGFR4-specific CARs induced lysis of FGFR4-postiveRH30 cells, but not FGFR4-negative Raji cells.

FIG. 12 is a graph showing interferon (IFN)-γ release induced by T cellsexpressing the FGFR4-specific M410 long, M412 short and M412 long CARs.IFN-γ released by RH30 (FGFR4+), SKES1 (FGFR4+), Raji (FGFR4-) and K562(FGFR4-) cells is shown.

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing areshown using standard three letter code for amino acids, as defined in 37C.F.R. 1.822. The Sequence Listing is submitted as an ASCII text file,created on Jun. 15, 2021, 104 KB, which is incorporated by referenceherein. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of the V_(H) of the BT53 mouseanti-FGFR4 mAb.

SEQ ID NO: 2 is the amino acid sequence of the V_(L) of the BT53 mouseanti-FGFR4 mAb.

SEQ ID NO: 3 is the amino acid sequence of the V_(H) of the 3A11 mouseanti-FGFR4 mAb.

SEQ ID NO: 4 is the amino acid sequence of the V_(L) of the 3A11 mouseanti-FGFR4 mAb.

SEQ ID NO: 5 is the amino acid sequence of the V_(H) of the 1G5 mouseanti-FGFR4 mAb.

SEQ ID NO: 6 is the amino acid sequence of the V_(L) of the 1G5 mouseanti-FGFR4 mAb.

SEQ ID NO: 7 is the amino acid sequence of the V_(H) of the 29.2 rabbitanti-FGFR4 mAb.

SEQ ID NO: 8 is the amino acid sequence of the V_(L) of the 29.2 rabbitanti-FGFR4 mAb.

SEQ ID NO: 9 is the amino acid sequence of the V_(H) of the 57.1 rabbitanti-FGFR4 mAb.

SEQ ID NO: 10 is the amino acid sequence of the V_(L) of the 57.1 rabbitanti-FGFR4 mAb.

SEQ ID NO: 11 is the amino acid sequence of the V_(H) of the M408 humananti-FGFR4 scFv.

SEQ ID NO: 12 is the amino acid sequence of the V_(L) of the M408 humananti-FGFR4 scFv.

SEQ ID NO: 13 is the amino acid sequence of the V_(H) of the M409 humananti-FGFR4 scFv.

SEQ ID NO: 14 is the amino acid sequence of the V_(L) of the M409 humananti-FGFR4 scFv.

SEQ ID NO: 15 is the amino acid sequence of the V_(H) of the M410 humananti-FGFR4 scFv.

SEQ ID NO: 16 is the amino acid sequence of the V_(L) of the M410 humananti-FGFR4 scFv.

SEQ ID NO: 17 is the amino acid sequence of the V_(H) of the M412 humananti-FGFR4 scFv.

SEQ ID NO: 18 is the amino acid sequence of the V_(L) of the M412 humananti-FGFR4 scFv.

SEQ ID NO: 19 is the amino acid sequence of the V_(H) of the M414 humananti-FGFR4 scFv.

SEQ ID NO: 20 is the amino acid sequence of the V_(L) of the M414 humananti-FGFR4 scFv.

SEQ ID NO: 21 is the amino acid sequence of the V_(H) of the M415 humananti-FGFR4 scFv.

SEQ ID NO: 22 is the amino acid sequence of the V_(L) of the M415 humananti-FGFR4 scFv.

SEQ ID NO: 23 is the amino acid sequence of the V_(H) of the M417 humananti-FGFR4 scFv.

SEQ ID NO: 24 is the amino acid sequence of the V_(L) of the M417 humananti-FGFR4 scFv.

SEQ ID NO: 25 is the amino acid sequence of the V_(H) of the M418 humananti-FGFR4 scFv.

SEQ ID NO: 26 is the amino acid sequence of the V_(L) of the M418 humananti-FGFR4 scFv.

SEQ ID NO: 27 is the amino acid sequence of the V_(H) of the M422 humananti-FGFR4 scFv.

SEQ ID NO: 28 is the amino acid sequence of the V_(L) of the M422 humananti-FGFR4 scFv.

SEQ ID NO: 29 is the amino acid sequence of the V_(H) of the M424 humananti-FGFR4 scFv.

SEQ ID NO: 30 is the amino acid sequence of the V_(L) of the M424 humananti-FGFR4 scFv.

SEQ ID NO: 31 is the amino acid sequence of a peptide linker forantibody-based CARs.

SEQ ID NO: 32 is the amino acid sequence of a peptide linker featured inscFv sequences.

SEQ ID NO: 33 is the amino acid sequence of an alternative peptidelinker featured in scFv sequences.

SEQ ID NO: 34 is the amino acid sequence of an exemplary signal peptide.

SEQ ID NO: 35 is a short linker domain for Ig binding domains totransmembrane sequences (short spacer).

SEQ ID NO: 36 is a linker domain composed of 2 Ig C domains (CH2CH3)used to link Ig binding domains to transmembrane sequences (longspacer).

SEQ ID NO: 37 is the amino acid sequence of a scFv including the V_(H)and the V_(L) of the BT53 mouse anti-FGFR4 mAb.

SEQ ID NO: 38 is the amino acid sequence of a scFv including the V_(H)and the V_(L) of the 3A11 mouse anti-FGFR4 mAb.

SEQ ID NO: 39 is the amino acid sequence of a scFv including the V_(H)and the V_(L) of the 1G5 mouse anti-FGFR4 mAb.

SEQ ID NO: 40 is the amino acid sequence of a scFv including thehumanized V_(H) and the V_(L) of the BT53 mouse anti-FGFR4 mAb.

SEQ ID NO: 41 is the amino acid sequence of a scFv including thehumanized V_(H) and the V_(L) of the 3A11 mouse anti-FGFR4 mAb.

SEQ ID NO: 42 is the amino acid sequence of a scFv including thehumanized V_(H) and the V_(L) of the 1G5 mouse anti-FGFR4 mAb.

SEQ ID NO: 43 is the amino acid sequence of a scFv including the V_(H)and V_(L) of the 29.2 rabbit anti-FGFR4 mAb.

SEQ ID NO: 44 is the amino acid sequence of a scFv including the V_(H)and V_(L) of the 57.1 rabbit anti-FGFR4 mAb.

SEQ ID NO: 45 is the amino acid sequence of a scFv including thehumanized V_(H) and V_(L) of the 29.2 rabbit anti-FGFR4 mAb.

SEQ ID NO: 46 is the amino acid sequence of a scFv including thehumanized V_(H) and V_(L) of the 57.1 rabbit anti-FGFR4 mAb.

SEQ ID NO: 47 is the amino acid sequence of the M408 human anti-FGFR4scFv.

SEQ ID NO: 48 is the amino acid sequence of the M409 human anti-FGFR4scFv.

SEQ ID NO: 49 is the amino acid sequence of the M410 human anti-FGFR4scFv.

SEQ ID NO: 50 is the amino acid sequence of the M412 human anti-FGFR4scFv.

SEQ ID NO: 51 is the amino acid sequence of the M414 human anti-FGFR4scFv.

SEQ ID NO: 52 is the amino acid sequence of the M415 human anti-FGFR4scFv.

SEQ ID NO: 53 is the amino acid sequence of the M417 human anti-FGFR4scFv.

SEQ ID NO: 54 is the amino acid sequence of the M418 human anti-FGFR4scFv.

SEQ ID NO: 55 is the amino acid sequence of the M422 human anti-FGFR4scFv.

SEQ ID NO: 56 is the amino acid sequence of the M424 human anti-FGFR4scFv.

SEQ ID NO: 57 is the amino acid sequence of an exemplary CD28transmembrane domain.

SEQ ID NO: 58 is the amino acid sequence of an exemplary CD28 signalingdomain.

SEQ ID NO: 59 is the amino acid sequence of exemplary CD28 transmembraneand signaling domains.

SEQ ID NO: 60 is the amino acid sequence of an exemplary CD8transmembrane domain.

SEQ ID NO: 61 is the amino acid sequence of an exemplary CD8 extendedtransmembrane domain.

SEQ ID NO: 62 is the amino acid sequence of an exemplary CD137 signalingdomain.

SEQ ID NO: 63 is the amino acid sequence of an exemplary CD137 signalingdomain.

SEQ ID NO: 64 is the amino acid sequence of an exemplary CD3 zetasignaling domain.

SEQ ID NO: 65 is the amino acid sequence of the transmembrane andintracellular domains of an exemplary second generation CAR including aCD28 transmembrane domain and a CD3 zeta signaling domain (“28z”).

SEQ ID NO: 66 is the amino acid sequence of the transmembrane andintracellular domains of an exemplary second generation CAR including aCD8 transmembrane domain, CD137 (4-1BB) signaling domain, and a CD3 zetasignaling domain (“BBz”).

SEQ ID NO: 67 is the amino acid sequence of the transmembrane andintracellular domains of an exemplary third generation CAR including aCD8 transmembrane domain, a CD28 signaling domain, a CD137 (4-1BB)signaling domain, and a CD3 zeta signaling domain (“28BBz”).

SEQ ID NO: 68 is the amino acid sequence of the 29.2 L CAR.

SEQ ID NO: 69 is the amino acid sequence of the 29.2 CAR.

SEQ ID NO: 70 is the amino acid sequence of the 57.1 L CAR.

SEQ ID NO: 71 is the amino acid sequence of the 57.1 CAR.

DETAILED DESCRIPTION I. Abbreviations

-   -   ADC antibody-drug conjugate    -   ADCC antibody-dependent cell-mediated cytotoxicity    -   ARMS alveolar rhabdomyosarcoma    -   CAR chimeric antigen receptor    -   CDC complement-dependent cytotoxicity    -   CDR complementarity determining region    -   CTL cytotoxic T lymphocyte    -   DMDM duocarmycin DM    -   ECD extracellular domain    -   ELISA enzyme-linked immunosorbent assay    -   ERMS embryonal rhabdomyosarcoma    -   Fc constant fragment    -   FGFR4 fibroblast growth factor receptor 4    -   IHC immunohistochemistry    -   ITAM immunoreceptor tyrosine-based activation motif    -   mAb monoclonal antibody    -   MMAF monomethyl auristatin F    -   PBD pyrrolobenzodiazepine    -   PBMC peripheral blood mononuclear cell    -   PE Pseudomonas exotoxin    -   RMS rhabdomyosarcoma    -   scFv single chain variable fragment    -   TMA tissue microarray    -   VH variable heavy    -   VL variable light

II. Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Anti-microtubule agent: A drug that interferes with microtubules.Anti-microtubule agents block cell growth by stopping mitosis.

Anti-mitotic agent: A drug or compound that blocks mitosis.

Antibody: A polypeptide ligand comprising at least a light chain and/orheavy chain immunoglobulin variable region which recognizes and binds(such as specifically recognizes and specifically binds) an epitope ofan antigen, such as FGFR4, or a fragment thereof. Immunoglobulinmolecules are composed of a heavy and a light chain, each of which has avariable region, termed the variable heavy (V_(H)) region and thevariable light (V_(L)) region. Together, the V_(H) region and the V_(L)region are responsible for binding the antigen recognized by theantibody.

Antibodies include intact immunoglobulins and the variants and portions(fragments) of antibodies well known in the art, such as single-domainantibodies (e.g. V_(H) domain antibodies), Fab fragments, Fab′fragments, F(ab)′2 fragments, single chain Fv proteins (“scFv”), anddisulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusionprotein in which a light chain variable region of an immunoglobulin anda heavy chain variable region of an immunoglobulin are bound by alinker, while in dsFvs, the chains have been mutated to introduce adisulfide bond to stabilize the association of the chains. The term“antibody” also includes genetically engineered forms such as chimericantibodies (for example, humanized murine antibodies) andheteroconjugate antibodies (such as bispecific antibodies). See also,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby, J., Immunology, 3^(rd) Ed., W. H. Freeman & Co., New York,1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (κ). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The amino acid sequenceboundaries of a given CDR can be readily determined using any of anumber of well-known schemes, including those described by Kabat et al.(Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991; the “Kabat” numbering scheme), Chothiaet al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia etal., Nature 342:877, 1989; and Al-Lazikani et al., (JMB 273,927-948,1997; the “Chothia” numbering scheme), and the ImMunoGeneTics (IMGT)database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT”numbering scheme). The Kabat and IMGT databases are maintained online.The sequences of the framework regions of different light or heavychains are relatively conserved within a species, such as humans. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and areoften identified by the chain in which the particular CDR is located.Thus, a V_(H) CDR3 (or HCDR3) is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1(or LCDR1) is the CDR1 from the variable domain of the light chain ofthe antibody in which it is found. An antibody that binds FGFR4, forexample, will have a specific V_(H) region and the V_(L) regionsequence, and thus specific CDR sequences. Antibodies with differentspecificities (i.e. different combining sites for different antigens)have different CDRs. Although it is the CDRs that vary from antibody toantibody, only a limited number of amino acid positions within the CDRsare directly involved in antigen binding. These positions within theCDRs are called specificity determining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and/or heavy chain genesof a single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds FGFR4.

A “human” antibody (also called a “fully human” antibody) is an antibodythat includes human framework regions and all of the CDRs from a humanimmunoglobulin. In one example, the framework and the CDRs are from thesame originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. A “humanized” immunoglobulin is animmunoglobulin including a human framework region and one or more CDRsfrom a non-human (for example a mouse, rabbit, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor,” and the human immunoglobulin providing the frameworkis termed an “acceptor.” In one embodiment, all the CDRs are from thedonor immunoglobulin in a humanized immunoglobulin. Constant regionsneed not be present, but if they are, they must be substantiallyidentical to human immunoglobulin constant regions, i.e., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences. A “humanized antibody” is an antibody comprising a humanizedlight chain and a humanized heavy chain immunoglobulin. A humanizedantibody binds to the same antigen as the donor antibody that providesthe CDRs. The acceptor framework of a humanized immunoglobulin orantibody may have a limited number of substitutions by amino acids takenfrom the donor framework. Humanized or other monoclonal antibodies canhave additional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. Humanized immunoglobulins can be constructed by means ofgenetic engineering (see for example, U.S. Pat. No. 5,585,089).

Antibody-drug conjugate (ADC): A molecule that includes an antibody (orantigen-binding fragment of an antibody) conjugated to a drug, such as acytotoxic agent. ADCs can be used to specifically target a drug tocancer cells through specific binding of the antibody to a tumor antigenexpressed on the cell surface. Exemplary drugs for use with ADCs includeanti-microtubule agents (such as maytansinoids, auristatin E andauristatin F) and interstrand crosslinking agents (e.g.,pyrrolobenzodiazepines; PDB s).

Anti-microtubule agent: A type of drug that blocks cell growth bystopping mitosis. Anti-microtubule agents, also referred to as“anti-mitotic agents,” are used to treat cancer.

Binding affinity: Affinity of an antibody for an antigen. In oneembodiment, affinity is calculated by a modification of the Scatchardmethod described by Frankel et al. (Mol. Immunol., 16:101-106, 1979). Inanother embodiment, binding affinity is measured by an antigen/antibodydissociation rate. In another embodiment, binding affinity is measuredby a competition radioimmunoassay. In another embodiment, bindingaffinity is measured by ELISA. An antibody that “specifically binds” anantigen (such as FGFR4) is an antibody that binds the antigen with highaffinity and does not significantly bind other unrelated antigens.

Breast cancer: A type of cancer that forms in the tissues of the breast,typically in the ducts and lobules. In some embodiments, a patient withbreast cancer is node-positive, meaning the breast cancer has spread tothe lymph nodes.

Chemotherapeutic agent: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth, such as psoriasis. In oneembodiment, a chemotherapeutic agent is an agent of use in treating aFGFR4-positive cancer, such as rhabdomyosarcoma, lung cancer, livercancer, breast cancer, pancreatic cancer and prostate cancer. In oneembodiment, a chemotherapeutic agent is a radioactive compound. One ofskill in the art can readily identify a chemotherapeutic agent of use(see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter86 in Harrison's Principles of Internal Medicine, 14th edition; Perry etal., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., ©2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.):Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-YearBook, 1995; Fischer, D. S., Knobf, M. F., Durivage, H. J. (eds): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).Combination chemotherapy is the administration of more than one agent totreat cancer. One example is the administration of an antibody (orimmunoconjugate or ADC) that binds FGFR4 used in combination with aradioactive or chemical compound.

Chimeric antigen receptor (CAR): A chimeric molecule that includes anantigen-binding portion (such as a monoclonal antibody or fragmentthereof) and a signaling domain, such as a signaling domain from a Tcell receptor (e.g. CD3ξ). Typically, CARs are comprised of a bindingmoiety (e.g. a scFv), a transmembrane domain and an endodomain. Theendodomain typically includes a signaling chain having an immunoreceptortyrosine-based activation motif (ITAM), such as CD3ξ or FcεRIγ. In someinstances, the endodomain further includes the intracellular portion ofat least one additional co-stimulatory domain, such as CD28 and/orCD137.

Conservative variant: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease the affinityof a protein, such as an antibody to FGFR4. For example, a monoclonalantibody that specifically binds FGFR4 can include at most about 1, atmost about 2, at most about 5, at most about 10, or at most about 15conservative substitutions and specifically bind a FGFR4 polypeptide.The term “conservative variant” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatthe antibody specifically binds FGFR4. Non-conservative substitutionsare those that reduce an activity or binding to FGFR4.

Complementarity determining region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of an Igeach have three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2and HCDR3, respectively.

Contacting: Placement in direct physical association; includes both insolid and liquid form.

Cytotoxic agent: Any drug or compound that kills cells.

Degenerate variant: In the context of the present disclosure, a“degenerate variant” refers to a polynucleotide encoding a FGFR4polypeptide or an antibody that binds FGFR4 that includes a sequencethat is degenerate as a result of the genetic code. There are 20 naturalamino acids, most of which are specified by more than one codon.Therefore, all degenerate nucleotide sequences are included as long asthe amino acid sequence of the FGFR4 polypeptide or antibody that bindsFGFR4 encoded by the nucleotide sequence is unchanged.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, cancer. Diagnostic methodsdiffer in their sensitivity and specificity. The “sensitivity” of adiagnostic assay is the percentage of diseased individuals who testpositive (percent of true positives). The “specificity” of a diagnosticassay is one minus the false positive rate, where the false positiverate is defined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis. “Prognostic” is theprobability of development (e.g., severity) of a pathologic condition,such as cancer or metastasis.

Drug: Any compound used to treat, ameliorate or prevent a disease orcondition in a subject. In some embodiments herein, the drug is ananti-cancer agent, for example a cytotoxic agent, such as ananti-mitotic or anti-microtubule agent.

Duocarmycin: A cytotoxic small molecule that induces cell death bybinding to the minor groove of DNA and alkylating the adenine.

Effector molecule: The portion of an antibody conjugate (orimmunoconjugate) that is intended to have a desired effect on a cell towhich the conjugate is targeted. Effector molecules are also known aseffector moieties (EMs), therapeutic agents, diagnostic agents, orsimilar terms. Therapeutic agents (or drugs) include such compounds assmall molecules, nucleic acids, proteins, peptides, amino acids orderivatives, glycoproteins, radioisotopes, lipids, carbohydrates, orrecombinant viruses. Nucleic acid therapeutic and diagnostic moietiesinclude antisense nucleic acids, derivatized oligonucleotides forcovalent cross-linking with single or duplex DNA, and triplex formingoligonucleotides. Alternatively, the effector molecule can be containedwithin an encapsulation system, such as a liposome or micelle, which isconjugated to the antibody. Encapsulation shields the effector moleculefrom direct exposure to the circulatory system. Means of preparingliposomes attached to antibodies are well known to those of skill in theart (see, for example, U.S. Pat. No. 4,957,735; and Connor et al., PharmTher 28:341-365, 1985). Diagnostic agents or moieties includeradioisotopes and other detectable labels (e.g., fluorophores,chemiluminescent agents, and enzymes). Radioactive isotopes include ³⁵S,¹¹C, ¹³N, ¹⁵O, ¹⁸F, ¹⁹F, ^(99m)Tc, ¹³¹I, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹Inand ¹²⁵I.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide, such as FGFR4.

Fibroblast growth factor receptor (FGFR): A family of tyrosine kinasereceptors activated by fibroblast growth factors (FGF), comprisingextracellular immunoglobulin-like domains, a transmembrane domain, andan intracellular tyrosine kinase domain. The family includes at leastfour members: FGFR1, FGFR2, FGFR3, and FGFR4.

Framework region: Amino acid sequences interposed between CDRs.Framework regions include variable light and variable heavy frameworkregions. The framework regions serve to hold the CDRs in an appropriateorientation for antigen binding.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4⁺ response or a CD8⁺ response. In another embodiment, theresponse is a B cell response, and results in the production ofantigen-specific antibodies.

Immunoliposome: A liposome with antibodies or antibody fragmentsconjugated to its surface. Immunoliposomes can carry cytotoxic agents orother drugs to antibody-targeted cells, such as tumor cells.

Interstrand crosslinking agent: A type of cytotoxic drug capable ofbinding covalently between two strands of DNA, thereby preventing DNAreplication and/or transcription.

Isolated: An “isolated” biological component, such as a nucleic acid,protein (including antibodies) or organelle, has been substantiallyseparated or purified away from other biological components in theenvironment (such as a cell) in which the component naturally occurs,i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins andorganelles. Nucleic acids and proteins that have been “isolated” includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes. In one example, a “labeled antibody” refers to incorporationof another molecule in the antibody. For example, the label is adetectable marker, such as the incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionucleotides (such as ³⁵S, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ¹⁹F,^(99m)Tc, ¹³¹I, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In and ¹²⁵I), fluorescentlabels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanidephosphors), enzymatic labels (such as horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (such as a leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags), or magnetic agents, such as gadolinium chelates. In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

Linker: In some cases, a linker is a peptide within an antibody bindingfragment (such as an Fv fragment) which serves to indirectly bond thevariable heavy chain to the variable light chain. “Linker” can alsorefer to a peptide serving to link a targeting moiety, such as anantibody, to an effector molecule, such as a cytotoxin or a detectablelabel.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a radionuclide, drug or other molecule to apolypeptide, such as an antibody or antibody fragment. In the specificcontext, the terms include reference to joining a ligand, such as anantibody moiety, to an effector molecule. The linkage can be either bychemical or recombinant means. “Chemical means” refers to a reactionbetween the antibody moiety and the effector molecule such that there isa covalent bond formed between the two molecules to form one molecule.

Liver cancer: A type of cancer than forms in the tissues of the liver.Types of liver cancers include, for example, hepatocellular carcinoma(HCC), cholangiocarcinoma (also known as bile duct cancer), angiosarcomaand hepatoblastoma.

Lung cancer: Cancer that forms in tissues of the lung, usually in thecells lining air passages.

The two main types are small cell lung cancer and non-small cell lungcancer. These types are diagnosed based on how the cells look under amicroscope.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Neoplasia, malignancy, cancer or tumor: A neoplasm is an abnormal growthof tissue or cells that results from excessive cell division. Neoplasticgrowth can produce a tumor. The amount of a tumor in an individual isthe “tumor burden” which can be measured as the number, volume, orweight of the tumor. A tumor that does not metastasize is referred to as“benign.” A tumor that invades the surrounding tissue and/or canmetastasize is referred to as “malignant.”

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Pancreatic cancer: A disease in which malignant (cancer) cells are foundin the tissues of the pancreas. Also called exocrine cancer.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the antibodies and conjugates disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop,such as a reduction in tumor burden or a decrease in the number of sizeof metastases. “Ameliorating” refers to the reduction in the number orseverity of signs or symptoms of a disease, such as cancer.

Prostate Cancer: A malignant tumor, generally of glandular origin, ofthe prostate. Prostate cancers include adenocarcinomas and small cellcarcinomas. Many prostate cancers express prostate specific antigen(PSA).

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell. Inone embodiment, a preparation is purified such that the protein orpeptide represents at least 50% of the total peptide or protein contentof the preparation. Substantial purification denotes purification fromother proteins or cellular components. A substantially purified proteinis at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific,non-limiting example, a substantially purified protein is 90% free ofother proteins or cellular components.

Pyrrolobenzodiazepine (PBD): A class of sequence-selective DNAminor-groove binding crosslinking agents originally discovered inStreptomyces species. PDBs are significantly more potent than systemicchemotherapeutic drugs. The mechanism of action of PBDs is associatedwith their ability to form an adduct in the minor groove of DNA, therebyinterfering with DNA processing. In the context of the presentdisclosure, PBDs include naturally produced and isolated PBDs,chemically synthesized naturally occurring PBDs, and chemicallysynthesized non-naturally occurring PBDs. PBDs also include monomeric,dimeric and hybrid PBDs (for a review see Gerratana, Med Res Rev32(2):254-293, 2012).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor by the artificial manipulation of isolated segments of nucleic acids,for example, by genetic engineering techniques.

Rhabdomyosarcoma (RMS): A soft tissue malignant tumor of skeletal muscleorigin. The most common primary sites for rhabdomyosarcoma are the headand neck (e.g., parameningeal, orbit, pharyngeal, etc.), thegenitourinary tract, and the extremities. Other less common primarysites include the trunk, chest wall, the abdomen (including theretroperitoneum and biliary tract), and the perineal/anal region. Thereare at least two types of RMS; the most common forms are alveolar RMS(ARMS) and embryonal histological RMS (ERMS). Approximately 20% ofchildren with rhabdomyosarcoma have the ARMS subtype. An increasedfrequency of this subtype is noted in adolescents and in patients withprimary sites involving the extremities, trunk, and perineum/perianalregion. ARMS is associated with chromosomal translocations encoding afusion gene involving FKHR on chromosome 13 and members of the PAXfamily. The embryonal subtype is the most frequently observed subtype inchildren, accounting for approximately 60-70% of rhabdomyosarcomas ofchildhood. Tumors with embryonal histology typically arise in the headand neck region or in the genitourinary tract, although they may occurat any primary site. ERMS is characterized by a younger age atdiagnosis, loss of heterozygosity, and altered genomic imprinting.

Sample (or biological sample): A biological specimen containing genomicDNA, RNA (including mRNA), protein, or combinations thereof, obtainedfrom a subject. Examples include, but are not limited to, peripheralblood, tissue, cells, urine, saliva, tissue biopsy, fine needleaspirate, surgical specimen, and autopsy material. In one example, asample includes a tumor biopsy.

Sequence identity: The similarity between amino acid or nucleic acidsequences is expressed in terms of the similarity between the sequences,otherwise referred to as sequence identity. Sequence identity isfrequently measured in terms of percentage identity (or similarity orhomology); the higher the percentage, the more similar the two sequencesare. Homologs or variants of a polypeptide or nucleic acid molecule willpossess a relatively high degree of sequence identity when aligned usingstandard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds FGFR4 or a fragment thereof are typicallycharacterized by possession of at least about 75%, for example at leastabout 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted overthe full length alignment with the amino acid sequence of the antibodyusing the NCBI Blast 2.0, gapped blastp set to default parameters. Forcomparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). When aligning short peptides (fewerthan around 30 amino acids), the alignment should be performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Proteins with evengreater similarity to the reference sequences will show increasingpercentage identities when assessed by this method, such as at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% sequence identity. When less than the entire sequence is beingcompared for sequence identity, homologs and variants will typicallypossess at least 80% sequence identity over short windows of 10-20 aminoacids, and may possess sequence identities of at least 85% or at least90% or 95% depending on their similarity to the reference sequence.Methods for determining sequence identity over such short windows areavailable at the NCBI website on the internet. One of skill in the artwill appreciate that these sequence identity ranges are provided forguidance only; it is entirely possible that strongly significanthomologs could be obtained that fall outside of the ranges provided.

Small molecule: A molecule, typically with a molecular weight less thanabout 1000 Daltons, or in some embodiments, less than about 500 Daltons,wherein the molecule is capable of modulating, to some measurableextent, an activity of a target molecule.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

Therapeutically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit or suppress growthof a tumor. In one embodiment, a therapeutically effective amount is theamount necessary to eliminate, reduce the size, or prevent metastasis ofa tumor. When administered to a subject, a dosage will generally be usedthat will achieve target tissue concentrations (for example, in tumors)that has been shown to achieve a desired in vitro effect.

Toxin: An agent that directly or indirectly inhibits the growth ofand/or kills cells. Toxins include, for example, Pseudomonas exotoxin(PE, such as PE35, PE37, PE38 and PE40), diphtheria toxin (DT),botulinum toxin, abrin, ricin, saporin, restrictocin or gelonin, ormodified toxins thereof. For example, PE and DT are highly toxiccompounds that typically bring about death through liver toxicity. PEand DT, however, can be modified into a form for use as an immunotoxinby removing the native targeting component of the toxin (such as domainIa of PE or the B chain of DT) and replacing it with a differenttargeting moiety, such as an antibody.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. “Comprising A or B” means including A, or B, or Aand B. It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

III. Monoclonal Antibodies Specific for FGFR4

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma ofchildhood. Two major subtypes, embryonal RMS (ERMS) and alveolar RMS(ARMS), harbor distinct cytogenetic and molecular abnormalities, some ofwhich are associated with poor prognosis. The FGFR4 gene is amplified,overexpressed, and mutationally activated in human RMS, and as such is atumor-specific target. Previous studies of FGFR4 indicate that this geneplays a role in tumorigenesis and is crucial for the survival,proliferation, metastasis and drug-resistance of RMS. High expression ofFGFR4 has been associated with an aggressive phenotype and poorsurvival. Conversely, genetic or pharmacologic inhibition of FGFR4signaling has been found to inhibit tumor growth in vitro and in vivo.

Disclosed herein are monoclonal antibodies, or antigen-binding fragmentsthereof, that specifically bind fibroblast growth factor receptor 4(FGFR4). The antibodies were selected from mice and rabbits immunizedwith the extracellular domain of human FGFR4 (hFGFR4-ECD), and from ahuman scFv library. Chimeric antigen receptors, antibody-drugconjugates, immunoconjugates, bispecific antibodies, immunoliposomes andcompositions comprising the FGFR4-specific antibodies are also disclosedherein. The monoclonal antibodies and antibody compositions can be usedto diagnose or treat a FGFR4-positive cancer, such as rhabdomyosarcoma,lung cancer, liver cancer, breast cancer, pancreatic cancer or prostatecancer.

Three mouse monoclonal antibodies (BT53, 3A11 and 1G5), two rabbitmonoclonal antibodies (29.2 and 57.1) and 10 human scFv (M408, M409,M410, M412, M414, M415, M417, M418, M422 and M424) that specificallybind FGFR4 were identified. The CDR sequences of the antibodiesdisclosed herein were determined using IMGT. However, one of skill inthe art could readily determine the CDR boundaries using alternativenumbering schemes, including the Kabat or Chothia numbering schemes.

Mouse Monoclonal Antibody Sequences

The VH and VL domain sequences of mouse monoclonal antibodies BT53, 3A11and 1G5 and provided below. The CDR sequences, as determined by IMGT,are shown in bold.

SEQ ID NO: 1 is the amino acid sequence of the V_(H)of the BT53 mouse anti-FGFR4 mAb:QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKKIPVYGLEWIGAIDPETYGTAYNQKFKGKATLTADKSSSTAYMEVRSLTSEDSAVYYCTRGGYY GSDFDYWGQGTTLTVSSSEQ ID NO: 2 is the amino acid sequence of the V_(L)of the BT53 mouse anti-FGFR4 mAb:NIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSDPYTFGGGT KLEIKSEQ ID NO: 3 is the amino acid sequence of the V_(H)of the 3A11 mouse anti-FGFR4 mAb:QVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYY GSDYDYWGQGTTLTVSSSEQ ID NO: 4 is the amino acid sequence of the V_(L)of the 3A11 mouse anti-FGFR4 mAb:DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQT FGGGTKLEIKSEQ ID NO: 5 is the amino acid sequence of the V_(H)of the 1G5 mouse anti-FGFR4 mAb:QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKKIPVYGLEWIGAIDPETYGTAYNQKFKGKATLTADKSSSTAYMEVRSLTSEDSAVYYCTRGGYY GSDFDYWGQSEQ ID NO: 6 is the amino acid sequence of the V_(L)of the 1G5 mouse anti-FGFR4 mAb:DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPWTFGGGT KLEIK

Rabbit Monoclonal Antibody Sequences

The VH and VL domain sequences of rabbit monoclonal antibodies 29.2 and57.1 are provided below. The CDR sequences, as determined by IMGT, areshown in bold.

SEQ ID NO: 7 is the amino acid sequence of the V_(H)of the 29.2 rabbit anti-FGFR4 mAb:QSVKESEGRLVTPGTPLTLTCTVSGFSLSSNSVGWVRQAPGKGLEWIGIISSSGNRYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCGGDPVSWYGD IWGPGTLVTVSSSEQ ID NO: 8 is the amino acid sequence of the V_(L)of the 29.2 rabbit anti-FGFR4 mAb:LLVTSLLLCELPHPAFLLIPDTELVLTQTPSSVSAAVGGTVTINCQSSPSLYKNNYLSWYQQKPGQPPKLLIYSASTLASGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCLGGYSLSSDSPRAFGGGTEVVVKSEQ ID NO: 9 is the amino acid sequence of the V_(H)of the 57.1 rabbit anti-FGFR4 mAb:QSVKESEGRLVTPGTPLTLTCTVSGFSLSTYAMSWVRQAPEKGLEWIGIIYATAETYYATWARGRFTISKTSTTVDLKITSPATEDTATYFCARLNGDGSGT YAYDIWGPGTLVTVSSSEQ ID NO: 10 is the amino acid sequence of the V_(L)of the 57.1 rabbit anti-FGFR4 mAb:LLVTSLLLCELPHPAFLLIPDTELVMTQTPSPVSAAVGGTVTINCQASQSISSSYLSWYQQKPGQPPKLLIYKASTRPSGVSSRFKGSGSGTQFTLTISGVQCADAATYYCLYGYYIDSGADNSFGGGTEVVVKHuman scFv Sequences

Provided below are the amino acid sequences of the VH and VL domains ofhuman scFv M408, M409, M410, M412, M414, M415, M417, M418, M422 andM424. The CDR sequences, as determined by IMGT, are shown in bold.

SEQ ID NO: 11 is the amino acid sequence of the V_(H)of the M408 human anti-FGFR4 scFv:EVQLVQSGVEGKKPEAPVKVSCKASGYTFTNYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARDPVL LWDGMDVWGQGTTSEQ ID NO: 12 is the amino acid sequence of the V_(L)of the M408 human anti-FGFR4 scFv.DIQMTQSPSSLSASVGDRVTITCRASQTISRYLNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFTLTISSLQPEDFATYFCQQTYSPPITFGQGT RLEIKRSEQ ID NO: 13 is the amino acid sequence of the V_(H)of the M409 human anti-FGFR4 scFv.AAQAAQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDASKNQFSLQLNSVTPEDTAVY YCSGSYSTFDIWGQGTMSEQ ID NO: 14 is the amino acid sequence of the V_(L)of the M409 human anti-FGFR4 scFv.NFMLTQPHSVSGSPGKTVTLSCTCSGGNIADAYVQWYQQRPGSAPRIVIYEDKQRPSGVPDRFSGSIDSSSNSASLTISGLRTEDEADYYCQSYDTNNFWVF GGGTKLTVLGSEQ ID NO: 15 is the amino acid sequence of the V_(H)of the M410 human anti-FGFR4 scFv.QVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASTIPY YGDYVEDYYGMDVWGQGTTSEQ ID NO: 16 is the amino acid sequence of the V_(L)of the M410 human anti-FGFR4 scFv.NFMLTQPHSVSESPGRTVSISCTRGSGSIADDYVQWYQQRPGGSPTIVIYEDNQRPSGVPDRFSGSIDTSSNSASLTISGLTTEDEAVYYCQSYDYRDHWVF GGGTQLTVLGSEQ ID NO: 17 is the amino acid sequence of the V_(H)of the M412 human anti-FGFR4 scFv.QAAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSTYYADSVKGRFTMSRDNSKNTLYLQMNSLRAEDTAVYYCARVG LQSGAFDIWGQGTTSEQ ID NO: 18 is the amino acid sequence of the V_(L)of the M412 human anti-FGFR4 scFv.DIQMTQSPSSLSASVGDRVTITCQASQDIYTYLNWYQQKPGKAPMLVIHDTSNLETGAPSRFSGGGSGTDFSFTISSLQPEDFATYYCQQYDALPFTFGQGT KLEIKRSEQ ID NO: 19 is the amino acid sequence of the V_(H)of the M414 human anti-FGFR4 scFv.EVQLVQFGAEVKKPGSSVKVSCKASGGTFSSYAISWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPGG EGLGAIDGFDIWGQGTTSEQ ID NO: 20 is the amino acid sequence of the V_(L)of the M414 human anti-FGFR4 scFv.DIQMTQSPSSLSASVGDRVTIACRASQTISRYLNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFTLTISSLQPEDFATYFCQQTYSPPITFGQGT RLEIKRSEQ ID NO: 21 is the amino acid sequence of the V_(H)of the M415 human anti-FGFR4 scFv.QVQLVESGGGVVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAWPE YSSSADAFDIWGQGTMSEQ ID NO: 22 is the amino acid sequence of the V_(L)of the M415 human anti-FGFR4 scFv.DIQLTQSPSSLSASVGDRVTITCQASQDIDNYLNWFQQKPGKPPKLLISDASSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNFPITFGQGT KLEIKRGQAGQGPDKTSEQ ID NO: 23 is the amino acid sequence of the V_(H)of the M417 human anti-FGFR4 scFv.EVQLVESGGALVQPGGSLRLSCAASGFTFTNYGIIWVRQAPGKGPEWVSGVSGNAVHTYYADSVKGRFTISRDNSKNMVYLQMNSLRSDDTAVYYCARGWDL DYWGQGTLSEQ ID NO: 24 is the amino acid sequence of the V_(L)of the M417 human anti-FGFR4 scFv.EIVLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKWYDASNLETGVPSRFIGGGSGTDFTLTISSLQPEDFATYYCQQHDSLPLSFGGGTKL EIKRSEQ ID NO: 25 is the amino acid sequence of the V_(H)of the M418 human anti-FGFR4 scFv.QLQLQESGPGLVKPSETLSLTCVVFDYSISSGYYWGWIRQPPGKGLEWIGSINYSGNTYYNPSLKSRVTISVDTSKNQFSLNLRSVTAADTAVYYCARSVDT APGFDYWGQGTLSEQ ID NO: 26 is the amino acid sequence of the V_(L)of the M418 human anti-FGFR4 scFv.DIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGT KLDIKRSEQ ID NO: 27 is the amino acid sequence of the V_(H)of the M422 human anti-FGFR4 scFv.EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATERAV AGPGAFDIWGQGTMSEQ ID NO: 28 is the amino acid sequence of the V_(L)of the M422 human anti-FGFR4 scFv.EIVLTQSPSSLSASVGDRVTIACRASQTISRYLNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFTLTISSLQPEDFATYFCQQTYSPPITFGQGT RLEIKRSEQ ID NO: 29 is the amino acid sequence of the V_(H)of the M424 human anti-FGFR4 scFv.QVQLVETGGGVVQPGTSLRLSCAGSGFTFSESGMHWVRQAPGKGLEWMALILNDGISNFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSLGG NGAFDIWGQGTMSEQ ID NO: 30 is the amino acid sequence of the V_(L)of the M424 human anti-FGFR4 scFv.DIQLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLEIGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHDNLPLSFGGGT KLDIKR

TABLE 1 IMGT CDR Sequences of FGFR4 Specific Antibodies SEQ ID A.A.SEQ ID A.A. BT53 NO: 1 Sequence NO: 2 Sequence HCDR1 26-33 GYTFTDYELCDR1 27-32 ENVVTY HCDR2 51-58 IDPETYGT LCDR2 50-52 GAS HCDR3  96-109CTRGGYYGSDF LCDR3 88-98 CGQGYSDPYTF DYW SEQ ID A.A. SEQ ID A.A. 3AllNO: 3 Sequence NO: 4 Sequence HCDR1 26-33 GYTFTDYE LCDR1 27-33 QSLLDSDHCDR2 51-58 IDPETGGT LCDR2 55-57 LVS HCDR3  96-109 CTRGNYYGSDY LCDR3 93-103 CWQGTHFPQTF DYW SEQ ID A.A. SEQ ID A.A. IG5 NO: 5 Sequence NO: 6Sequence HCDR1 26-33 GYTFTDYE LCDR1 27-32 QNINVW HCDR2 52-59 IDPETYGTLCDR2 50-52 KAS HCDR3  98-111 CTRGGYYGSD LCDR3 88-98 CQQGQSYPWTF FDYWSEQ ID A.A. SEQ ID A.A. 29.2 NO: 7 Sequence NO: 8 Sequence HCDR1 25-32GFSLSSNS LCDR1 49-56 PSLYKNNY HCDR2 50-56 ISSSGNR LCDR2 74-76 SAS HCDR3 92-104 CGGDPVSWYGD LCDR3 112-126 CLGGYSLSSDS IW PRAF SEQ ID A.A. SEQ IDA.A. 57.1 NO: 9 Sequence NO: 10 Sequence HCDR1 25-32 GFSLSTYA LCDR149-55 QSISSSY HCDR2 50-56 IYATAET LCDR2 73-75 KAS HCDR3  92-108CARLNGDGSG LCDR3 111-125 CLYGYYIDSGA TYAYDIW DNSF SEQ ID A.A. SEQ IDA.A. M408 NO: 11 Sequence NO: 12 Sequence HCDR1 26-33 GYTFTNYY LCDR127-32 QTISRY HCDR2 51-58 VDPEDGET LCDR2 50-52 AAS HCDR3  96-110CARDPVLLWD LCDR3 88-97 CQQTYSPPITF GMDVW SEQ ID A.A. SEQ ID A.A. M409NO: 13 Sequence NO: 14 Sequence HCDR1 31-40 GDSVSSNSAA LCDR1 26-33GGNIADAY HCDR2 58-66 TYYRSKWYN LCDR2 51-53 EDK HCDR3 104-114 CSGSYSTFDLCDR3  91-102 CQSYDTNNFW IW VF SEQ ID A.A. SEQ ID A.A. M410 NO: 15Sequence NO: 16 Sequence HCDR1 26-33 GGTFSSYA LCDR1 26-33 SGSIADDY HCDR251-58 IIPIFGTA LCDR2 51-53 EDN HCDR3  96-116 CASTIPYYGDY LCDR3  91-102CQSYDYRDHW VEDYYGMDVW VF SEQ ID A.A. SEQ ID A.A. M412 NO: 17 SequenceNO: 18 Sequence HCDR1 29-36 GFTFSSYA LCDR1 27-33 QDIYTYL HCDR2 54-60IYSGGST LCDR2 50-53 DTS HCDR3  98-111 CARVGLQSGA LCDR3 89-99 CQQYDALPFTFFDIW SEQ ID A.A. SEQ ID A.A. M414 NO: 19 Sequence NO: 20 Sequence HCDR126-33 GGTFSSYA LCDR1 37-32 QTISRY HCDR2 51-58 VDPEDGET LCDR2 51-53 AASHCDR3  96-114 CARDPGGEGLG LCDR3 89-99 CQQTYSPPITF AIDGFDIW SEQ ID A.A.SEQ ID A.A. M415 NO: 21 Sequence NO: 22 Sequence HCDR1 26-33 GFTFSSYALCDR1 27-32 QDIDNY HCDR2 51-58 ISYDGSNK LCDR2 50-52 DAS HCDR3  96-113CARAWPEYSSS LCDR3 88-98 CQQYDNFPITF ADAFDIW SEQ ID A.A. SEQ ID A.A. M417NO: 23 Sequence NO: 24 Sequence HCDR1 26-33 GFTFTNYG LCDR1 27-32 QDISNYHCDR2 51-58 VSGNAVHT LCDR2 50-52 DAS HCDR3  97-106 CARGWDLDYW LCDR388-98 CQQHDSLPLSF SEQ ID A.A. SEQ ID A.A. M418 NO: 25 Sequence NO: 26Sequence HCDR1 26-34 DYSISSGYY LCDR1 27-32 QGISSY HCDR2 52-58 INYSGNTLCDR2 50-52 DAS HCDR3  96-109 CARSVDTAPG LCDR3 88-98 CQQYDNLPLTF FDYWSEQ ID A.A. SEQ ID A.A. M422 NO: 27 Sequence NO: 28 Sequence HCDR1 27-34GYTFTDYY LCDR1 27-32 QTISRY HCDR2 52-59 VDPEDGET LCDR2 50-52 AAS HCDR3 96-111 CATERAVAGP LCDR3 88-98 CQQTYSPPITF GAFDIW SEQ ID A.A. SEQ IDA.A. M424 NO: 29 Sequence NO: 30 Sequence HCDR1 26-33 GFTFSESG LCDR127-32 QDISNY HCDR2 51-58 ILNDGISN LCDR2 50-52 DAS HCDR3  96-109CASSLGGNGA LCDR3 88-98 CQQHDNLPLSF FDIW

Peptide Linker and Signal Peptide Sequences

SEQ ID NO: 31 is the amino acid sequence of apeptide linker for antibody-based CARs: GGGGSGGGGSGGGGSSEQ ID NO: 32 is the amino acid sequence of apeptide linker featured in scFv sequences: VTVSSGGGGSGGGASSGGGSSEQ ID NO: 33 is the amino acid sequence of analternative peptide linker featured in scFv sequences:VTVSSGGGGSGGGASGGGGS SEQ ID NO: 34 is the amino acid sequence of anexemplary signal peptide: LLVTSLLLCELPHPAFLLIPDTSEQ ID NO: 35 is a short linker domain for Igbinding domains to transmembrane sequences (short spacer):KTTPPSVYGRVKDPKAAAIE SEQ ID NO: 36 is a linker domain composed of 2 Ig Cdomains (CH2CH3) used to link Ig binding domains totransmembrane sequences (long spacer):EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKAAAIEscFv Sequences

Provided below are the amino acid sequences of exemplary scFv. CDRsequences, as determined by IMGT, are shown in bold; linker sequencesare shown in italics.

SEQ ID NO: 37 is the amino acid sequence of a scFv including theV_(H )and the V_(L )of the BT53 mouse anti-FGFR4 mAb:QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKKIPVYGLEWIGAIDPETYGTAYNQKFKGKATLTADKSSSTAYMEVRSLTSEDSAVYYCTRGGYYGSDFDYWGQGTTLTVSSGGGGSGGGGSGGGGSNIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSDPYTFGGGTKLEIKSEQ ID NO: 38 is the amino acid sequence of a scFv including theV_(H )and the V_(L )of the 3A11 mouse anti-FGFR4 mAb:QVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYYGSDYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKSEQ ID NO: 39 is the amino acid sequence of a scFv including theV_(H )and the V_(L )of the 1G5 mouse anti-FGFR4 mAb:QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKKIPVYGLEWIGAIDPETYGTAYNQKFKGKATLTADKSSSTAYMEVRSLTSEDSAVYYCTRGGYYGSDFDYWGQGGGGSGGGGSGGGGSDIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPWTFGGGTKLEIKSEQ ID NO: 40 is the amino acid sequence of a scFv including thehumanized V_(H )and the V_(L )of the BT53 mouse anti-FGFR4 mAb:QVQLQQSGAEVKKPGSSVKVSCKASGYTFTDYEISWVRQAPGQGLEWMGGIDPETYGTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTRGGYYGSDFDYWWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASENVVTYAWYQQKPGQAPRLLIYGASRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCGQGYSDPYTFFGQGTKLEIKRSEQ ID NO: 41 is the amino acid sequence of a scFv including thehumanized V_(H )and the V_(L )of the 3A11 mouse anti-FGFR4 mAb:QVQLQQSGAEVKKPGSSVKVSCKASGYTFTDYEISWVRQAPGQGLEWMGGIDPETGGTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTRGNYYGSDYDYWWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSLLDSDAWYQQKPGQAPRLLIYLVSRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCWQGTHFPQTFFGQGTKLEIKRSEQ ID NO: 42 is the amino acid sequence of a scFv including thehumanized V_(H )and the V_(L )of the 1G5 mouse anti-FGFR4 mAb:QVQLQQSGAEVKKPGSSVKVSCKASGYTFTDYEISWVRQAPGQGLEWMGGIDPETYGTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTRGGYYGSDFDYWWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQNINVWAWYQQKPGQAPRLLIYKASRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGQSYPWTFFGQGTKLEIKRSEQ ID NO: 43 is the amino acid sequence of a scFv including theVH and VL of the 29.2 rabbit anti-FGFR4 mAb:QSVKESEGRLVTPGTPLTLTCTVSGFSLSSNSVGWVRQAPGKGLEWIGIISSSGNRYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCGGDPVSWYGDIWGPGTLVTVSSGGGGSGGGGSGGGGSLLVTSLLLCELPHPAFLLIPDTELVLTQTPSSVSAAVGGTVTINCQSSPSLYKNNYLSWYQQKPGQPPKLLIYSASTLASGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCLGGYSLSSDSPRAFGGGTEVVVKSEQ ID NO: 44 is the amino acid sequence of a scFv including theV_(H )and V_(L )of the 57.1 rabbit anti-FGFR4 mAb:QSVKESEGRLVTPGTPLTLTCTVSGFSLSTYAMSWVRQAPEKGLEWIGIIYATAETYYATWARGRFTISKTSTTVDLKITSPATEDTATYFCARLNGDGSGTYAYDIWGPGTLVTVSSGGGGSGGGGSGGGGSLLVTSLLLCELPHPAFLLIPDTELVMTQTPSPVSAAVGGTVTINCQASQSISSSYLSWYQQKPGQPPKLLIYKASTRPSGVSSRFKGSGSGTQFTLTISGVQCADAATYYCLYGYYIDSGADNSFGGGTEVVVKSEQ ID NO: 45 is the amino acid sequence of a scFv including thehumanized V_(H )and V_(L )of the 29.2 rabbit anti-FGFR4 mAb:QVQLQQSGAEVKKPGSSVKVSCKASGFSLSSNSISWVRQAPGQGLEWMGGISSSGNRNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCGGDPVSWYGDIWWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASPSLYKNNYAWYQQKPGQAPRLLIYSASRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCLGGYSLSSDSPRAFFGQGTKLEIKRSEQ ID NO: 46 is the amino acid sequence of a scFv including thehumanized V_(H )and V_(L )of the 57.1 rabbit anti-FGFR4 mAb:QVQLQQSGAEVKKPGSSVKVSCKASGFSLSTYAISWVRQAPGQGLEWMGGIYATAETNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLNGDGSGTYAYDIWWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSISSSYAWYQQKPGQAPRLLIYKASRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCLYGYYIDSGADNSFFGQGTKLEIKRSEQ ID NO: 47 is the amino acid sequence of the M408 humananti-FGFR4 scFv:EVQLVQSGVEGKKPEAPVKVSCKASGYTFTNYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARDPVLLWDGMDVWGQGTTVTVSSGGGGSGGGASSGGGSDIQMTQSPSSLSASVGDRVTITCRASQTISRYLNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFTLTISSLQPEDFATYFCQQTYSPPITFGQGTRLEIKRSEQ ID NO: 48 is the amino acid sequence of the M409 humananti-FGFR4 scFv:AAQAAQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDASKNQFSLQLNSVTPEDTAVYYCSGSYSTFDIWGQGTMVTVSSGGGGSGGGASSGGGSNFMLTQPHSVSGSPGKTVTLSCTCSGGNIADAYVQWYQQRPGSAPRIVIYEDKQRPSGVPDRFSGSIDSSSNSASLTISGLRTEDEADYYCQSYDTNNFWVFGGGTKLTVL GSEQ ID NO: 49 is the amino acid sequence of the M410 humananti-FGFR4 scFv:QVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASTIPYYGDYVEDYYGMDVWGQGTTVTVSSGGGGSGGGASSGGGSNFMLTQPHSVSESPGRTVSISCTRGSGSIADDYVQWYQQRPGGSPTIVIYEDNQRPSGVPDRFSGSIDTSSNSASLTISGLTTEDEAVYYCQSYDYRDHWVFGGGTQLTV LGSEQ ID NO: 50 is the amino acid sequence of the M412 humananti-FGFR4 scFv:QAAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSTYYADSVKGRFTMSRDNSKNTLYLQMNSLRAEDTAVYYCARVGLQSGAFDIWGQGTTVTVSSGGGGSGGGASGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDIYTYLNWYQQKPGKAPMLVIHDTSNLETGAPSRFSGGGSGTDFSFTISSLQPEDFATYYCQQYDALPFTFGQGTKLEIKRSEQ ID NO: 51 is the amino acid sequence of the M414 humananti-FGFR4 scFv:EVQLVQFGAEVKKPGSSVKVSCKASGGTFSSYAISWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPGGEGLGAIDGFDIWGQGTTVTVSSGGGGSGGGASGGGGSDIQMTQSPSSLSASVGDRVTIACRASQTISRYLNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFTLTISSLQPEDFATYFCQQTYSPPITFGQGTRLEIKRSEQ ID NO: 52 is the amino acid sequence of the M415 humananti-FGFR4 scFv:QVQLVESGGGVVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAWPEYSSSADAFDIWGQGTMVTVSSGGGGSGGGASGGGGSDIQLTQSPSSLSASVGDRVTITCQASQDIDNYLNWFQQKPGKPPKLLISDASSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNFPITFGQGTKLEIKRGQAGQG PDKTSEQ ID NO: 53 is the amino acid sequence of the M417 humananti-FGFR4 scFv:EVQLVESGGALVQPGGSLRLSCAASGFTFTNYGIIWVRQAPGKGPEWVSGVSGNAVHTYYADSVKGRFTISRDNSKNMVYLQMNSLRSDDTAVYYCARGWDLDYWGQGTLVTVSSGGGGSGGGASGGGGSEIVLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFIGGGSGTDFTLTISSLQPEDFATYYCQQHDSLPLSFGGGTKLEIKRSEQ ID NO: 54 is the amino acid sequence of the M418 humananti-FGFR4 scFv:QLQLQESGPGLVKPSETLSLTCVVFDYSISSGYYWGWIRQPPGKGLEWIGSINYSGNTYYNPSLKSRVTISVDTSKNQFSLNLRSVTAADTAVYYCARSVDTAPGFDYWGQGTLVTVSSGGGGSGGGASSGGGSDIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKLDIKRSEQ ID NO: 55 is the amino acid sequence of the M422 humananti-FGFR4 scFv:EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATERAVAGPGAFDIWGQGTMVTVSSGGGGSGGGASGGGGSEIVLTQSPSSLSASVGDRVTIACRASQTISRYLNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFTLTISSLQPEDFATYFCQQTYSPPITFGQGTRLEIKRSEQ ID NO: 56 is the amino acid sequence of the M424 humananti-FGFR4 scFv:QVQLVETGGGVVQPGTSLRLSCAGSGFTFSESGMHWVRQAPGKGLEWMALILNDGISNFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSLGGNGAFDIWGQGTMVTVSSGGGGSGGGASSGGGSDIQLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLEIGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHDNLPLSFGGGTKLDIKR

Signaling Domains

Amino acid sequences of exemplary signaling domains that can be used forchimeric antigen receptors (CARs) are provided below.

SEQ ID NO: 57 is the amino acid sequence of anexemplary CD28 transmembrane domain:IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSEQ ID NO: 58 is the amino acid sequence of anexemplary CD28 signaling domain:SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSSEQ ID NO: 59 is the amino acid sequence ofexemplary CD28 transmembrane and signaling domains:IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSSEQ ID NO: 60 is the amino acid sequence of anexemplary CD8 transmembrane domain:TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCSEQ ID NO: 61 is the amino acid sequence of anexemplary CD8 extended transmembrane domain:FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSEQ ID NO: 62 is the amino acid sequence of anexemplary CD137 signaling domain:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSEQ ID NO: 63 is the amino acid sequence of anexemplary CD137 signaling domain:RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSEQ ID NO: 64 is the amino acid sequence of anexemplary CD3 zeta signaling domain:RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPRSEQ ID NO: 65 is the amino acid sequence of thetransmembrane and intracellular domains of anexemplary second generation CAR including a CD28transmembrane domain and a CD3 zeta signaling domain (“28z”):AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPRSEQ ID NO: 66 is the amino acid sequence of thetransmembrane and intracellular domains of anexemplary 2^(nd )generation CAR including a CD8transmembrane domain, CD137 (4-1BB) signalingdomain, and a CD3 zeta signaling domain (“BBz”):AAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 67 is the amino acid sequence of thetransmembrane and intracellular domains of anexemplary 3rd generation CAR including a CD8transmembrane domain, a CD28 signaling domain,a CD137 (4-1BB) signaling domain, and a CD3 zetasignaling domain (“28BBz”):AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Exemplary CARs

Listed below are exemplary FGFR4-specific CARs that can be used foradoptive immunotherapy. All the CAR molecules include an N-terminalsignal peptide (SP; SEQ ID NO: 34), a scFv sequence, a transmembrane(TM) sequence and a CD3 zeta signaling sequence. CARs that refer to “SH”do not have a CH2CH3 spacer domain.

The extracellular domain of the following exemplary CARs is a “short”extracellular domain, which is a CAR without a CH2CH3 spacer:

BT53 SP-murine BT53 scFv 3A11 SP-murine 3A11 scFv 1G5 SP-murine 1G5 scFv29.2 SP-rabbit 29.2 scFv 57.1 SP-rabbit 57.1 scFv hBT53 SP-humanizedBT53 scFv h3A11 SP-humanized 3A11 scFv h1G5 SP-humanized 1G5 scFv h29.2SP-humanized 29.2 scFv h57.1 SP-humanized 57.1 scFv M408 SP-M408 scFvM409 SP-M409 scFv M410 SP-M410 scFv M412 SP-M412 scFv M414 SP-M414 scFvM415 SP-M415 scFv M417 SP-M417 scFv M418 SP-M418 scFv M422 SP-M422 scFvM424 SP-M424 scFv

The extracellular domain of the following exemplary CARs is a “long” CARextracellular domain with a CH2CH3 spacer (SEQ ID: NO 36, CH2CH3):

BT53L SP-murine BT53 scFv-CH2CH3 3A11L SP-murine 3A11 scFv-CH2CH3 1G5LSP-murine 1G5 scFv-CH2CH3 29.2L SP-rabbit 29.2 scFv-CH2CH3 57.1LSP-rabbit 57.1 scFv-CH2CH3 hBT53L SP-humanized BT53 scFv-CH2CH3 h3A11LSP-humanized 3A11 scFv-CH2CH3 h1G5L SP-humanized 1G5 scFv-CH2CH3 h29.2LSP-humanized 29.2 scFv-CH2CH3 h57.1L SP-humanized 57.1 scFv-CH2CH3 M408LSP-M408 scFv-CH2CH3 M409L SP-M409 scFv-CH2CH3 M410L SP-M410 scFv-CH2CH3M412L SP-M412 scFv-CH2CH3 M414L SP-M414 scFv-CH2CH3 M415L SP-M415scFv-CH2CH3 M417L SP-M417 scFv-CH2CH3 M418L SP-M418 scFv-CH2CH3 M422LSP-M422 scFv-CH2CH3 M424L SP-M424 scFv-CH2CH3

For the CAR transmembrane domain and intracellular domains, thefollowing nomenclature is used:

28z CD28 transmembrane—CD28 signaling—CD3 zeta signaling

BBz CD8 transmembrane—4-1BB/CD137 signaling—CD3 zeta signaling

28BBz CD8 transmembrane—CD28 signaling—4-1BB/CD137 signaling—CD3 zetasignaling

In some embodiments in which the CAR extracellular domains are notlinked to a CD8 transmembrane domain (i.e. 28z), a short linker (SEQ IDNO: 35) follows the scFv prior to joining the transmembrane sequence.

Exemplary CAR Sequences

The amino acid sequences of four exemplary CARs are provided below.Shown in italics are scFv peptide linkers; the long (CH2CH3) and shortlinkers for linking Ig domains to transmembrane domains are shown inbold.

29.2L (SEQ ID NO: 68):LLVTSLLLCELPHPAFLLIPDTELVLTQTPSSVSAAVGGTVTINCQSSPSLYKNNYLSWYQQKPGQPPKLLIYSASTLASGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCLGGYSLSSDSPRAFGGGTEVVVKGGGGSGGGGSGGGGSQSVKESEGRLVTPGTPLTLTCTVSGFSLSSNSVGWVRQAPGKGLEWIGIISSSGNRYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCGGDPVSWYGDIWGPGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK 29.2 (SEQ ID NO: 69):LLVTSLLLCELPHPAFLLIPDTELVLTQTPSSVSAAVGGTVTINCQSSPSLYKNNYLSWYQQKPGQPPKLLIYSASTLASGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCLGGYSLSSDSPRAFGGGTEVVVKGGGGSGGGGSGGGGSQSVKESEGRLVTPGTPLTLTCTVSGFSLSSNSVGWVRQAPGKGLEWIGIISSSGNRYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCGGDPVSWYGDIWGPGTLVTVSSKTTPPSVYGRVKDPK 57.1L (SEQ ID NO: 70):LLVTSLLLCELPHPAFLLIPDTELVMTQTPSPVSAAVGGTVTINCQASQSISSSYLSWYQQKPGQPPKLLIYKASTRPSGVSSRFKGSGSGTQFTLTISGVQCADAATYYCLYGYYIDSGADNSFGGGTEVVVKGGGGSGGGGSGGGGSQSVKESEGRLVTPGTPLTLTCTVSGFSLSTYAMSWVRQAPEKGLEWIGIIYATAETYYATWARGRFTISKTSTTVDLKITSPATEDTATYFCARLNGDGSGTYAYDIWGPGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK 57.1 (SEQ ID NO: 71):LLVTSLLLCELPHPAFLLIPDTELVMTQTPSPVSAAVGGTVTINCQASQSISSSYLSWYQQKPGQPPKLLIYKASTRPSGVSSRFKGSGSGTQFTLTISGVQCADAATYYCLYGYYIDSGADNSFGGGTEVVVKGGGGSGGGGSGGGGSQSVKESEGRLVTPGTPLTLTCTVSGFSLSTYAMSWVRQAPEKGLEWIGIIYATAETYYATWARGRFTISKTSTTVDLKITSPATEDTATYFCARLNGDGSGTYAYDIWGPGTLVTVSSKTTPPSVYGRVKDPK

Provided herein are isolated monoclonal antibodies that bind FGFR4, orantigen-binding fragments thereof, comprising a variable heavy (VH)domain and a variable light (VL) domain. In some embodiments, themonoclonal antibodies or antigen-binding fragments comprise at least aportion of one of the amino acid sequences set forth herein as SEQ IDNOs: 1-30, such as one or more (such as all three) CDR sequences fromone of SEQ ID NOs: 1-30. In some examples, the CDR locations aredetermined IMGT, Kabat or Chothia.

In some embodiments, the VH domain of the antibody comprises the CDRsequences of SEQ ID NO: 1 and the VL domain of the antibody comprisesthe CDR sequences of SEQ ID NO: 2; the VH domain of the antibodycomprises the CDR sequences of SEQ ID NO: 3 and the VL domain of theantibody comprises the CDR sequences of SEQ ID NO: 4; the VH domain ofthe antibody comprises the CDR sequences of SEQ ID NO: 5 and the VLdomain of the antibody comprises the CDR sequences of SEQ ID NO: 6; theVH domain of the antibody comprises the CDR sequences of SEQ ID NO: 7and the VL domain of the antibody comprises the CDR sequences of SEQ IDNO: 8; the VH domain of the antibody comprises the CDR sequences of SEQID NO: 9 and the VL domain of the antibody comprises the CDR sequencesof SEQ ID NO: 10; the VH domain of the antibody comprises the CDRsequences of SEQ ID NO: 11 and the VL domain of the antibody comprisesthe CDR sequences of SEQ ID NO: 12; the VH domain of the antibodycomprises the CDR sequences of SEQ ID NO: 13 and the VL domain of theantibody comprises the CDR sequences of SEQ ID NO: 14; the VH domain ofthe antibody comprises the CDR sequences of SEQ ID NO: 15 and the VLdomain of the antibody comprises the CDR sequences of SEQ ID NO: 16; theVH domain of the antibody comprises the CDR sequences of SEQ ID NO: 17and the VL domain of the antibody comprises the CDR sequences of SEQ IDNO: 18; the VH domain of the antibody comprises the CDR sequences of SEQID NO: 19 and the VL domain of the antibody comprises the CDR sequencesof SEQ ID NO: 20; the VH domain of the antibody comprises the CDRsequences of SEQ ID NO: 21 and the VL domain of the antibody comprisesthe CDR sequences of SEQ ID NO: 22; the VH domain of the antibodycomprises the CDR sequences of SEQ ID NO: 23 and the VL domain of theantibody comprises the CDR sequences of SEQ ID NO: 24; the VH domain ofthe antibody comprises the CDR sequences of SEQ ID NO: 25 and the VLdomain of the antibody comprises the CDR sequences of SEQ ID NO: 26; theVH domain of the antibody comprises the CDR sequences of SEQ ID NO: 27and the VL domain of the antibody comprises the CDR sequences of SEQ IDNO: 28; or the VH domain of the antibody comprises the CDR sequences ofSEQ ID NO: 29 and the VL domain of the antibody comprises the CDRsequences of SEQ ID NO: 30. In some examples, the CDR sequences aredetermined using the IMGT, Kabat or Chothia numbering scheme.

In some embodiments, the VH domain of the antibody comprises residues26-33, 51-58 and 96-109 of SEQ ID NO: 1 and the VL domain of theantibody comprises residues 27-32, 50-52 and 88-98 of SEQ ID NO: 2; theVH domain of the antibody comprises residues 26-33, 51-58 and 96-109 ofSEQ ID NO: 3 and the VL domain of the antibody comprises residues 27-33,55-57 and 93-103 of SEQ ID NO: 4; the VH domain of the antibodycomprises residues 26-33, 52-59 and 98-111 of SEQ ID NO: 5 and the VLdomain of the antibody comprises residues 27-32, 50-52 and 88-98 of SEQID NO: 6; the VH domain of the antibody comprises residues 25-32, 50-56and 92-104 of SEQ ID NO: 7 and the VL domain of the antibody comprisesresidues 49-56, 74-76 and 112-126 of SEQ ID NO: 8; the VH domain of theantibody comprises residues 25-32, 50-56 and 92-108 of SEQ ID NO: 9 andthe VL domain of the antibody comprises residues 49-55, 73-75 and111-125 of SEQ ID NO: 10; the VH domain of the antibody comprisesresidues 26-33, 51-58 and 96-110 of SEQ ID NO: 11 and the VL domain ofthe antibody comprises residues 27-32, 50-52 and 88-97 of SEQ ID NO: 12;the VH domain of the antibody comprises residues 31-40, 58-66 and104-114 of SEQ ID NO: 13 and the VL domain of the antibody comprisesresidues 26-33, 51-53 and 91-102 of SEQ ID NO: 14; the VH domain of theantibody comprises residues 26-33, 51-58 and 96-116 of SEQ ID NO: 15 andthe VL domain of the antibody comprises residues 26-33, 51-53 and 91-102of SEQ ID NO: 16; the VH domain of the antibody comprises residues29-36, 54-60 and 98-111 of SEQ ID NO: 17 and the VL domain of theantibody comprises residues 27-33, 50-53 and 89-99 of SEQ ID NO: 18; theVH domain of the antibody comprises residues 26-33, 51-58 and 96-114 ofSEQ ID NO: 19 and the VL domain of the antibody comprises residues37-32, 51-53 and 89-99 of SEQ ID NO: 20; the VH domain of the antibodycomprises residues 26-33, 51-58 and 96-113 of SEQ ID NO: 21 and the VLdomain of the antibody comprises residues 27-32, 50-52 and 88-98 of SEQID NO: 22; the VH domain of the antibody comprises residues 26-33, 51-58and 97-106 of SEQ ID NO: 23 and the VL domain of the antibody comprisesresidues 27-32, 50-52 and 88-98 of SEQ ID NO: 24; the VH domain of theantibody comprises residues 26-34, 52-58 and 96-109 of SEQ ID NO: 25 andthe VL domain of the antibody comprises residues 27-32, 50-52 and 88-98of SEQ ID NO: 26; the VH domain of the antibody comprises residues27-34, 52-59 and 96-111 of SEQ ID NO: 27 and the VL domain of theantibody comprises residues 27-32, 50-52 and 88-98 of SEQ ID NO: 28; orthe VH domain of the antibody comprises residues 26-33, 51-58 and 96-109of SEQ ID NO: 29 and the VL domain of the antibody comprises residues27-32, 50-52 and 88-98 of SEQ ID NO: 30.

In some embodiments, the amino acid sequence of the VH domain is atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 andthe amino acid sequence of the VL domain is at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to SEQ ID NO: 2; the amino acid sequence of theVH domain is at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% identical to SEQID NO: 3 and the amino acid sequence of the VL domain is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical to SEQ ID NO: 4; the amino acidsequence of the VH domain is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 5 and the amino acid sequence of the VL domainis at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 6;the amino acid sequence of the VH domain is at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to SEQ ID NO: 7 and the amino acid sequence ofthe VL domain is at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98% or at least 99% identical toSEQ ID NO: 8; the amino acid sequence of the VH domain is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical to SEQ ID NO: 9 and the amino acidsequence of the VL domain is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 10; the amino acid sequence of the VH domain isat least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical to SEQ ID NO: 11 andthe amino acid sequence of the VL domain is at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to SEQ ID NO: 12; the amino acid sequence of theVH domain is at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% identical to SEQID NO: 13 and the amino acid sequence of the VL domain is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical to SEQ ID NO: 14; the amino acidsequence of the VH domain is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 15 and the amino acid sequence of the VL domainis at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 16;the amino acid sequence of the VH domain is at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to SEQ ID NO: 17 and the amino acid sequence ofthe VL domain is at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98% or at least 99% identical toSEQ ID NO: 18; the amino acid sequence of the VH domain is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical to SEQ ID NO: 19 and the amino acidsequence of the VL domain is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 20; the amino acid sequence of the VH domain isat least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical to SEQ ID NO: 21 andthe amino acid sequence of the VL domain is at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to SEQ ID NO: 22; the amino acid sequence of theVH domain is at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% identical to SEQID NO: 23 and the amino acid sequence of the VL domain is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical to SEQ ID NO: 24; the amino acidsequence of the VH domain is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 25 and the amino acid sequence of the VL domainis at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 26;the amino acid sequence of the VH domain is at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to SEQ ID NO: 27 and the amino acid sequence ofthe VL domain is at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98% or at least 99% identical toSEQ ID NO: 28; or the amino acid sequence of the VH domain is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% identical to SEQ ID NO: 29 and theamino acid sequence of the VL domain is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identical to SEQ ID NO: 30.

In some embodiments, the amino acid sequence of the VH domain comprisesSEQ ID NO: 1 and the amino acid sequence of the VL domain comprises SEQID NO: 2; the amino acid sequence of the VH domain comprises SEQ ID NO:3 and the amino acid sequence of the VL domain comprises SEQ ID NO: 4;the amino acid sequence of the VH domain comprises SEQ ID NO: 5 and theamino acid sequence of the VL domain comprises SEQ ID NO: 6; the aminoacid sequence of the VH domain comprises SEQ ID NO: 7 and the amino acidsequence of the VL domain comprises SEQ ID NO: 8; the amino acidsequence of the VH domain comprises SEQ ID NO: 9 and the amino acidsequence of the VL domain comprises SEQ ID NO: 10; the amino acidsequence of the VH domain comprises SEQ ID NO: 11 and the amino acidsequence of the VL domain comprises SEQ ID NO: 12; the amino acidsequence of the VH domain comprises SEQ ID NO: 13 and the amino acidsequence of the VL domain comprises SEQ ID NO: 14; the amino acidsequence of the VH domain comprises SEQ ID NO: 15 and the amino acidsequence of the VL domain comprises SEQ ID NO: 16; the amino acidsequence of the VH domain comprises SEQ ID NO: 17 and the amino acidsequence of the VL domain comprises SEQ ID NO: 18; the amino acidsequence of the VH domain comprises SEQ ID NO: 19 and the amino acidsequence of the VL domain comprises SEQ ID NO: 20; the amino acidsequence of the VH domain comprises SEQ ID NO: 21 and the amino acidsequence of the VL domain comprises SEQ ID NO: 22; the amino acidsequence of the VH domain comprises SEQ ID NO: 23 and the amino acidsequence of the VL domain comprises SEQ ID NO: 24; the amino acidsequence of the VH domain comprises SEQ ID NO: 25 and the amino acidsequence of the VL domain comprises SEQ ID NO: 26; the amino acidsequence of the VH domain comprises SEQ ID NO: 27 and the amino acidsequence of the VL domain comprises SEQ ID NO: 28; or the amino acidsequence of the VH domain comprises SEQ ID NO: 29 and the amino acidsequence of the VL domain comprises SEQ ID NO: 30.

In some examples, antigen-binding fragment that binds FGFR4 is an Fabfragment, an Fab′ fragment, an F(ab)′2 fragment, a single chain variablefragment (scFv) or a disulfide stabilized variable fragment (dsFv).

In particular non-limiting examples, the fragment is a scFv comprisingan amino acid sequence at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 43or SEQ ID NO: 44. In non-limiting examples, the fragment is a scFvcomprising the amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 38, SEQID NO: 39, SEQ ID NO: 43 or SEQ ID NO: 44.

In particular non-limiting examples, the fragment is a humanized scFvcomprising an amino acid sequence at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identical to SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:45 or SEQ ID NO: 46. In specific examples, the fragment is a humanizedscFv comprising the amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 41,SEQ ID NO: 42, SEQ ID NO: 45 or SEQ ID NO: 46.

In some examples, the monoclonal antibody is an IgG. In other examples,the monoclonal antibody is an IgA, IgD, IgE or IgM.

In some embodiments, the antibody or antigen-binding fragment is a fullyhuman antibody or antigen-binding fragment. In other embodiments, theantibody or antigen-binding fragment is a chimeric, synthetic, humanizedor human antibody.

Further provided herein are antibody-drug conjugates (ADCs) that includea drug conjugated to a FGFR4 monoclonal antibody or antigen-bindingfragment disclosed herein. In some embodiments, the drug is a smallmolecule. In some embodiments, the drug is an anti-microtubule agent, ananti-mitotic agent and/or a cytotoxic agent. In particular examples, thedrug is monomethyl auristatin F (MMAF) or duocarmycin. ADCs are furtherdiscussed herein in section V below.

Also provided herein are chimeric antigen receptors (CARs) that includea monoclonal antibody or antigen-binding fragment disclosed herein, atransmembrane domain and a signaling domain. In some examples, the CARsfurther include a signal peptide and/or one or more linker peptides.

In some embodiments, the transmembrane domain of the CAR comprises aCD28 or a CD8 transmembrane domain. In some examples, the amino acidsequence of the CD28 transmembrane domain is at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to the amino acid sequence of SEQ ID NO: 57; orthe amino acid sequence of the CD8 transmembrane domain is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical to the amino acid sequence of SEQ IDNO: 60 or SEQ ID NO: 61. In specific non-limiting examples, the CD28transmembrane domain comprises or consists of the amino acid sequence ofSEQ ID NO: 57; or the CD8 transmembrane domain comprises or consists ofthe amino acid sequence of SEQ ID NO: 60 or SEQ ID NO: 61.

In some embodiments, the signaling domain of the CAR comprises a CD28,CD137 or CD3 signaling domain. In some examples, the amino acid sequenceof the CD28 signaling domain is at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identical to the amino acid sequence of SEQ ID NO: 58; the aminoacid sequence of the CD137 signaling domain is at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% identical to the amino acid sequence of SEQ ID NO:62 or SEQ ID NO: 63; or the amino acid sequence of the CD3ξ signalingdomain is at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% identical to theamino acid sequence of SEQ ID NO: 64. In specific non-limitingembodiments, the CD28 signaling domain comprises or consists of theamino acid sequence of SEQ ID NO: 58; the CD137 signaling domaincomprises or consists of the amino acid sequence of SEQ ID NO: 62 or SEQID NO: 63; or the CD3ξ signaling domain comprises or consists of theamino acid sequence of SEQ ID NO: 64.

In some examples, the amino acid sequence of the transmembrane andsignaling domains of the CAR is at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identical to the amino acid sequence of SEQ ID NO: 59, SEQ ID NO:65, SEQ ID NO: 66 or SEQ ID NO: 67. In particular examples, thetransmembrane and signaling domains of the CAR comprise or consist ofthe amino acid sequence of SEQ ID NO: 59, SEQ ID NO: 65, SEQ ID NO: 66or SEQ ID NO: 67.

In some examples, the amino acid sequence of the CAR is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical to SEQ ID NO: 68, SEQ ID NO: 69, SEQID NO: 70 or SEQ ID NO: 71. In particular examples, the amino acidsequence of the CAR comprises SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70 or SEQ ID NO: 71.

Also provided are isolated cells expressing a CAR disclosed herein. Insome embodiments, the cell is a cytotoxic T lymphocyte (CTL). CARs aredescribed further in section VI below.

Immunoconjugates that include a FGFR4-specific monoclonal antibody orantigen-binding fragment disclosed herein and an effector molecule arealso provided by the present disclosure. In some embodiments, theeffector molecule is a toxin, such as Pseudomonas exotoxin or a variantthereof. In other embodiments, the effector molecule is a detectablelabel, such as a fluorescent, radioactive or enzymatic label.Immunoconjugates are discussed in greater detail in section VIII below.

Bispecific antibodies that include a FGFR4-specific monoclonal antibodyor antigen-binding fragment disclosed herein and a second monoclonalantibody or antigen-binding fragment thereof are further provided. Insome embodiments, the second monoclonal antibody or antigen-bindingfragment thereof specifically binds a component of the T cell receptor,such as CD3, or specifically binds a natural killer (NK) cell activatingreceptor, such as CD16. In some examples, the FGFR4-specificantigen-binding fragment and the second antigen-binding fragment arescFv molecules. Bispecific antibodies are discussed in greater detail insection VII below.

Also provided herein are immunoliposomes that include a liposomeconjugated to a FGFR4-specific monoclonal antibody or antigen-bindingfragment disclosed herein. In some embodiments, the liposome comprises acytotoxic agent, such as an anti-cancer agent. Immunoliposomes arefurther described in section IX.

Further provided herein are compositions that include a disclosedFGFR4-speicific monoclonal antibody or antigen-binding fragment thereof,ADC, CAR, isolated cell, immunoconjugate, bispecific antibody orimmunoliposome and a pharmaceutically acceptable carrier. Compositionsand methods of their use are discussed further in section X below.

Also provided herein are isolated nucleic acid molecules encoding theFGFR4-specific monoclonal antibodies or antigen-binding fragments, CARs,immunoconjugates and bispecific antibodies disclosed herein. In someembodiments, the nucleic acid molecules are operably linked to apromoter. Further provided are vectors that include the nucleic acidmolecules disclosed herein. Isolated host cells transformed with thedisclosed nucleic acid molecules and vectors are further provided by thepresent disclosure.

Methods of inhibiting tumor growth or metastasis of a FGFR4-positivecancer are provided herein. In some embodiments, the method includesselecting a subject with a FGFR4-positive cancer and administering tothe subject a therapeutically effective amount of a FGFR4-specificmonoclonal antibody, antigen-binding fragment, ADC, CAR, isolated cell,immunoconjugate, bispecific antibody, immunoliposome or compositiondisclosed herein. Also provided are methods of treating a FGFR4-positivecancer in a subject by selecting a subject with a FGFR4-positive cancerand administering to the subject a therapeutically effective amount of amonoclonal antibody, antigen-binding fragment, ADC, CAR, isolated cell,immunoconjugate, bispecific antibody, immunoliposome or compositiondisclosed herein. In some examples, the FGFR4-positive cancer is arhabdomyosarcoma (RMS), lung cancer, liver cancer, breast cancer,pancreatic cancer or prostate cancer. In particular examples, the RMS isalveolar RMS (ARMS) or embryonal RMS (ERMS).

Further provided herein are methods of detecting expression of FGFR4 ina sample. In some embodiments, the method includes contacting the samplewith the monoclonal antibody or antigen-binding fragment disclosedherein; and detecting binding of the antibody or antigen-bindingfragment to the sample. In some examples, the monoclonal antibody orantigen-binding fragment is directly labeled. In other examples, themethod further includes contacting the monoclonal antibody orantigen-binding fragment with a second antibody (for example, ananti-IgG antibody), and detecting the binding of the second antibody tothe monoclonal antibody or antigen-binding fragment. In specificexamples, the sample is obtained from a subject suspected of having aFGFR4-positive cancer. The sample can be any suitable biologicalsamples, such as a cell or tissue sample. In some instances, the sampleis a tumor biopsy.

IV. Monoclonal Antibodies and Antigen-Binding Fragments Thereof

The monoclonal antibodies disclosed herein can be of any isotype. Themonoclonal antibody can be, for example, an IgM or an IgG antibody, suchas IgG₁ or an IgG₂. The class of an antibody that specifically bindsFGFR4 can be switched with another (for example, IgG can be switched toIgM), according to well-known procedures. Class switching can also beused to convert one IgG subclass to another, such as from IgG₁ to IgG₂.

Antibody fragments are also encompassed by the present disclosure, suchas single-domain antibodies (e.g., VH domain antibodies), Fab, F(ab′)₂,and Fv. These antibody fragments retain the ability to selectively bindwith the antigen. These antigen-binding fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains;

(5) Single chain antibody (such as scFv), a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule;

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of ascFv (also known as a “miniantibody”); and

(7) VH single-domain antibody, an antibody fragment consisting of theheavy chain variable domain.

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988).

In some cases, antibody fragments can be prepared by proteolytichydrolysis of the antibody or by expression in a host cell (such as E.coli) of DNA encoding the fragment. Antibody fragments can be obtainedby pepsin or papain digestion of whole antibodies by conventionalmethods. For example, antibody fragments can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′)2. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 3.5S Fab′monovalent fragments. Alternatively, an enzymatic cleavage using pepsinproduces two monovalent Fab′ fragments and an Fc fragment directly (seeU.S. Pat. Nos. 4,036,945 and 4,331,647).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules. Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and/or the V_(L)regions to increase yield. Conservative amino acid substitution tablesproviding functionally similar amino acids are well known to one ofordinary skill in the art. The following six groups are examples ofamino acids that are considered to be conservative substitutions for oneanother:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

V. Antibody-Drug Conjugates (ADCs)

ADCs are compounds comprised of a tumor antigen-specific antibody and adrug, typically a cytotoxic agent, such as an anti-microtubule agent orcross-linking agent. Because ADCs are capable of specifically targetingcancer cells, the drug can be much more potent than agents used forstandard chemotherapy. The most common cytotoxic drugs currently usedwith ADCs have an IC₅₀ that is 100- to 1000-fold more potent thanconventional chemotherapeutic agents. Common cytotoxic drugs includeanti-microtubule agents, such as maytansinoids and auristatins (such asauristatin E and auristatin F). Other cytotoxins for use with ADCsinclude pyrrolobenzodiazepines (PDBs), which covalently bind the minorgroove of DNA to form interstrand cros slinks. In many instances, ADCscomprise a 1:2 to 1:4 ratio of antibody to drug (Bander, ClinicalAdvances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

The antibody and drug can be linked by a cleavable or non-cleavablelinker. However, in some instances, it is desirable to have a linkerthat is stable in the circulation to prevent systemic release of thecytotoxic drug that could result in significant off-target toxicity.Non-cleavable linkers prevent release of the cytotoxic agent before theADC is internalized by the target cell. Once in the lysosome, digestionof the antibody by lysosomal proteases results in the release of thecytotoxic agent (Bander, Clinical Advances in Hematology & Oncology10(8; suppl 10):3-7, 2012).

One method for site-specific and stable conjugation of a drug to amonoclonal antibody is via glycan engineering. Monoclonal antibodieshave one conserved N-linked oligosaccharide chain at the Asn297 residuein the CH2 domain of each heavy chain (Qasba et al., Biotechnol Prog24:520-526, 2008). Using a mutant β1,4-galactosyltransferase enzyme(Y289 L-Gal-T1; U.S. Patent Application Publication Nos. 2007/0258986and 2006/0084162, herein incorporated by reference), 2-keto-galactose istransferred to free GlcNAc residues on the antibody heavy chain toprovide a chemical handle for conjugation.

The oligosaccharide chain attached to monoclonal antibodies can beclassified into three groups based on the terminal galactoseresidues—fully galactosylated (two galactose residues; IgG-G2), onegalactose residue (IgG-G1) or completely degalactosylated (IgG-G0).Treatment of a monoclonal antibody with β1,4-galactosidase converts theantibody to the IgG-G0 glycoform. The mutant β1,4-galactosyltransferaseenzyme is capable of transferring 2-keto-galactose or 2-azido-galactosefrom their respective UDP derivatives to the GlcNAc residues on theIgG-G1 and IgG-G0 glycoforms. The chemical handle on the transferredsugar enables conjugation of a variety of molecules to the monoclonalantibody via the glycan residues (Qasba et al., Biotechnol Prog24:520-526, 2008).

Provided herein are ADCs that include a drug (such as a cytotoxic agent)conjugated to a monoclonal antibody that binds (such as specificallybinds) FGFR4. In some embodiments, the drug is a small molecule. In someexamples, the drug is a cross-linking agent, an anti-microtubule agentand/or anti-mitotic agent, or any cytotoxic agent suitable for mediatingkilling of tumor cells. Exemplary cytotoxic agents include, but are notlimited to, a PDB, an auristatin, a maytansinoid, dolastatin,calicheamicin, nemorubicin and its derivatives, PNU-159682,anthracycline, vinca alkaloid, taxane, trichothecene, CC1065,camptothecin, elinafide, a combretastain, a dolastatin, a duocarmycin,an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, apuromycin, a tubulysin, a hemiasterlin, a spliceostatin, or apladienolide, as well as stereoisomers, isosteres, analogs, andderivatives thereof that have cytotoxic activity.

In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD).The natural product anthramycin (a PBD) was first reported in 1965(Leimgruber et al., J Am Chem Soc, 87:5793-5795, 1965; Leimgruber etal., J Am Chem Soc, 87:5791-5793, 1965). Since then, a number of PBDs,both naturally-occurring and synthetic analogues, have been reported(Gerratana, Med Res Rev 32(2):254-293, 2012; and U.S. Pat. Nos.6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and7,557,099). As one example, PDB dimers recognize and bind to specificDNA sequences, and have been shown to be useful as cytotoxic agents. PBDdimers have been conjugated to antibodies and the resulting ADC shown tohave anti-cancer properties (see, for example, US 2010/0203007).Exemplary linkage sites on the PBD dimer include the five-memberedpyrrolo ring, the tether between the PBD units, and the N10-C11 iminegroup (see WO 2009/016516; US 2009/304710; US 2010/047257; US2009/036431; US 2011/0256157; and WO 2011/130598).

In some embodiments, the ADC comprises an antibody conjugated to one ormore maytansinoid molecules. Maytansinoids are derivatives ofmaytansine, and are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos.4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; and 4,371,533.

In some embodiments, the ADC includes an antibody conjugated to adolastatin or auristatin, or an analog or derivative thereof (see U.S.Pat. Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatinsare derivatives of the marine mollusk compound dolastatin-10.Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al., Antimicrob Agents and Chemother 45(12):3580-3584, 2001)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al., Antimicrob Agents Chemother 42:2961-2965, 1998).Exemplary dolastatins and auristatins include, but are not limited to,dolastatin 10, auristatin E, auristatin F, auristatin EB (AEB),auristatin EFP (AEFP), MMAD (Monomethyl Auristatin D or monomethyldolastatin 10), MMAF (Monomethyl Auristatin F orN-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE(Monomethyl Auristatin E orN-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine),5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, forexample, U.S. Publication No. 2013/0129753).

In some embodiments, the ADC comprises an antibody conjugated to one ormore calicheamicin molecules. The calicheamicin family of antibiotics,and analogues thereof, are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations (Hinman et al., Cancer Res53:3336-3342, 1993; Lode et al., Cancer Res 58:2925-2928, 1998).Exemplary methods for preparing ADCs with a calicheamicin drug moietyare described in U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116; and5,767,285.

In some embodiments, the ADC comprises an anthracycline. Anthracyclinesare antibiotic compounds that exhibit cytotoxic activity. It is believedthat anthracyclines can operate to kill cells by a number of differentmechanisms, including intercalation of the drug molecules into the DNAof the cell thereby inhibiting DNA-dependent nucleic acid synthesis;inducing production of free radicals which then react with cellularmacromolecules to cause damage to the cells; and/or interactions of thedrug molecules with the cell membrane. Non-limiting exemplaryanthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin,daunorubicin, doxorubicin, epirubicin, nemorubicin, valrubicin andmitoxantrone, and derivatives thereof. For example, PNU-159682 is apotent metabolite (or derivative) of nemorubicin (Quintieri et al., ClinCancer Res 11(4):1608-1617, 2005). Nemorubicin is a semisynthetic analogof doxorubicin with a 2-methoxymorpholino group on the glycoside aminoof doxorubicin (Grandi et al., Cancer Treat Rev 17:133, 1990; Ripamontiet al., Br J Cancer 65:703-707, 1992).

In some embodiments, the ADC can further include a linker. In someexamples, the linker is a bifunctional or multifunctional moiety thatcan be used to link one or more drug moieties to an antibody to form anADC. In some embodiments, ADCs are prepared using a linker havingreactive functionalities for covalently attaching to the drug and to theantibody. For example, a cysteine thiol of an antibody can form a bondwith a reactive functional group of a linker or a drug-linkerintermediate to make an ADC.

In some examples, a linker has a functionality that is capable ofreacting with a free cysteine present on an antibody to form a covalentbond. Exemplary linkers with such reactive functionalities includemaleimide, haloacetamides, α-haloacetyl, activated esters such assuccinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates.

In some examples, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Examples ofsuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some cases, a heteroatom of the reactivefunctionality of the linker can react with an electrophilic group on anantibody and form a covalent bond to an antibody unit. Non-limitingexamples include hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate and arylhydrazide.

In some examples, the linker is a cleavable linker, which facilitatesrelease of the drug. Examples of cleavable linkers include acid-labilelinkers (for example, comprising hydrazone), protease-sensitive linkers(for example, peptidase-sensitive), photolabile linkers, anddisulfide-containing linkers (Chari et al., Cancer Res 52:127-131, 1992;U.S. Pat. No. 5,208,020).

The ADCs disclosed herein can be used for the treatment of aFGFR4-positive cancer alone or in combination with another therapeuticagent and/or in combination with any standard therapy for the treatmentof cancer (such as surgical resection of the tumor, chemotherapy orradiation therapy).

VI. Chimeric Antigen Receptors (CARs)

The disclosed monoclonal antibodies can also be used to produce CARs(also known as chimeric T cell receptors, artificial T cell receptors orchimeric immunoreceptors) and/or cytotoxic T lymphocytes (CTLs)engineered to express CARs. Generally, CARs include a binding moiety, anextracellular hinge and spacer element, a transmembrane region and anendodomain that performs signaling functions (Cartellieri et al., JBiomed Biotechnol 2010:956304, 2010). In many instances, the bindingmoiety is an antigen binding fragment of a monoclonal antibody, such asa scFv. Several different endodomains have been used to generate CARs.For example, the endodomain can consist of a signaling chain having anITAM, such as CD3ξ or FccRly. In some instances, the endodomain furtherincludes the intracellular portion of at least one additionalco-stimulatory domain, such as CD28 and/or CD137.

CTLs expressing CARs can be used to target a specific cell type, such asa tumor cell. Thus, the monoclonal antibodies disclosed herein can beused to engineer CTLs that express a CAR containing an antigen-bindingfragment of a FGFR4-specific antibody, thereby targeting the engineeredCTLs to FGFR4-expressing tumor cells. Engineered T cells have previouslybeen used for adoptive therapy for some types of cancer (see, forexample, Park et al., Mol Ther 15(4):825-833, 2007). The use of T cellsexpressing CARs is more universal than standard CTL-based immunotherapybecause CTLs expressing CARs are HLA unrestricted and can therefore beused for any patient having a tumor that expresses the target antigen.

Accordingly, provided herein are CARs that include a FGFR4-specificmonoclonal antibody, or antigen-binding fragment thereof, such as ascFv. Also provided are isolated nucleic acid molecules and vectorsencoding the CARs, and host cells, such as CTLs, expressing the CARs.CTLs expressing CARs comprised of a FGFR4-specific monoclonal antibody(or antibody binding fragment) can be used for the treatment of cancersthat express FGFR4, such as rhabdomyosarcoma, lung cancer, liver cancer,breast cancer, pancreatic cancer and prostate cancer.

VII. Bispecific Antibodies

Bispecific antibodies are recombinant proteins comprised ofantigen-binding fragments of two different monoclonal antibodies. Thus,bispecific antibodies bind two different antigens. Bispecific antibodiescan be used for cancer immunotherapy by simultaneously targeting, forexample, both CTLs (such as a CTL receptor component such as CD3) oreffector natural killer (NK) cells, and a tumor antigen. TheFGFR4-specific monoclonal antibodies disclosed herein can be used togenerate bispecific antibodies that target both FGFR4 and CTLs, ortargeting both FGFR4 and NK cells, thereby providing a means to treatFGFR4-expressing cancers.

Bi-specific T-cell engagers (BiTEs) are a type of bispecific monoclonalantibody that are fusions of a first single-chain variable fragment(scFv) that targets a tumor antigen and a second scFv that binds Tcells, such as bind CD3 on T cells.

Bi-specific killer cell engagers (BiKEs) are a type of bispecificmonoclonal antibody that are fusions of a first scFv that targets atumor antigen and a second scFv that binds a NK cell activatingreceptor, such as CD16.

Provided herein are bispecific monoclonal antibodies comprising aFGFR4-specific monoclonal antibody, or antigen-binding fragment thereof.In some embodiments, the bispecific monoclonal antibody furthercomprises a monoclonal antibody, or antigen-binding fragment thereof,that specifically binds a component of the T cell receptor, such as CD3.In other embodiments, the bispecific monoclonal antibody furthercomprises a monoclonal antibody, or antigen-binding fragment thereof,that specifically binds a NK cell activating receptor, such as CD16,Ly49, or CD94. In some examples, the antigen-binding fragments are scFv.Also provided are isolated nucleic acid molecules and vectors encodingthe bispecific antibodies, and host cells comprising the nucleic acidmolecules or vectors. Bispecific antibodies comprising a FGFR4-specificantibody, or antigen-binding fragment thereof, can be used for thetreatment of cancers that express FGFR4, such as rhabdomyosarcoma, lungcancer, liver cancer, breast cancer, pancreatic cancer and prostatecancer. Thus, provided herein are methods of treating a subject withcancer by selecting a subject with a cancer that expresses FGFR4, andadministering to the subject a therapeutically effective amount of theFGFR4-targeting bispecific antibody.

VIII. Immunoconjugates

The disclosed monoclonal antibodies specific for FGFR4 can be conjugatedto a therapeutic agent or effector molecule. Immunoconjugates include,but are not limited to, molecules in which there is a covalent linkageof a therapeutic agent to an antibody. A therapeutic agent is an agentwith a particular biological activity directed against a particulartarget molecule or a cell bearing a target molecule. One of skill in theart will appreciate that therapeutic agents can include various drugssuch as vinblastine, daunomycin and the like, cytotoxins such as nativeor modified Pseudomonas exotoxin or diphtheria toxin, encapsulatingagents (such as liposomes) that contain pharmacological compositions,radioactive agents such as ¹²⁵I, ³²P, ¹⁴C, ³H and ³⁵S and other labels,target moieties and ligands.

The choice of a particular therapeutic agent depends on the particulartarget molecule or cell, and the desired biological effect. Thus, forexample, the therapeutic agent can be a cytotoxin that is used to bringabout the death of a particular target cell (such as a tumor cell).Conversely, where it is desired to invoke a non-lethal biologicalresponse, the therapeutic agent can be conjugated to a non-lethalpharmacological agent or a liposome containing a non-lethalpharmacological agent.

With the therapeutic agents and antibodies described herein, one ofskill can readily construct a variety of clones containing functionallyequivalent nucleic acids, such as nucleic acids which differ in sequencebut which encode the same effector moiety or antibody sequence. Thus,the present disclosure provides nucleic acids encoding antibodies andconjugates and fusion proteins thereof.

Effector molecules can be linked to an antibody of interest using anynumber of means known to those of skill in the art. Both covalent andnoncovalent attachment means may be used. The procedure for attaching aneffector molecule to an antibody varies according to the chemicalstructure of the effector. Polypeptides typically contain a variety offunctional groups; such as carboxylic acid (COOH), free amine (—NH₂) orsulfhydryl (—SH) groups, which are available for reaction with asuitable functional group on an antibody to result in the binding of theeffector molecule. Alternatively, the antibody is derivatized to exposeor attach additional reactive functional groups. The derivatization mayinvolve attachment of any of a number of known linker molecules. Thelinker can be any molecule used to join the antibody to the effectormolecule. The linker is capable of forming covalent bonds to both theantibody and to the effector molecule. Suitable linkers are well knownto those of skill in the art and include, but are not limited to,straight or branched-chain carbon linkers, heterocyclic carbon linkers,or peptide linkers. Where the antibody and the effector molecule arepolypeptides, the linkers may be joined to the constituent amino acidsthrough their side groups (such as through a disulfide linkage tocysteine) or to the alpha carbon amino and carboxyl groups of theterminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, labels (such as enzymes or fluorescent molecules), drugs,toxins, and other agents to antibodies one skilled in the art will beable to determine a suitable method for attaching a given agent to anantibody or other polypeptide.

The antibodies or antibody fragments disclosed herein can be derivatizedor linked to another molecule (such as another peptide or protein). Ingeneral, the antibodies or portion thereof is derivatized such that thebinding to the target antigen is not affected adversely by thederivatization or labeling. For example, the antibody can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (for example, a bispecific antibody or a diabody), adetection agent, a pharmaceutical agent, and/or a protein or peptidethat can mediate association of the antibody or antibody portion withanother molecule (such as a streptavidin core region or a polyhistidinetag).

One type of derivatized antibody is produced by cross-linking two ormore antibodies (of the same type or of different types, such as tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (suchas disuccinimidyl suberate). Such linkers are commercially available.

The antibody can be conjugated with a detectable marker; for example, adetectable marker capable of detection by ELISA, spectrophotometry, flowcytometry, microscopy or diagnostic imaging techniques (such as computedtomography (CT), computed axial tomography (CAT) scans, magneticresonance imaging (MRI), nuclear magnetic resonance imaging NMRI),magnetic resonance tomography (MTR), ultrasound, fiberoptic examination,and laparoscopic examination). Specific, non-limiting examples ofdetectable markers include fluorophores, chemiluminescent agents,enzymatic linkages, radioactive isotopes and heavy metals or compounds(for example super paramagnetic iron oxide nanocrystals for detection byMRI). For example, useful detectable markers include fluorescentcompounds, including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, green fluorescent protein (GFP) and yellow fluorescentprotein (YFP). An antibody or antigen binding fragment can also beconjugated with enzymes that are useful for detection, such ashorseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase, glucose oxidase and the like. When an antibody or antigenbinding fragment is conjugated with a detectable enzyme, it can bedetected by adding additional reagents that the enzyme uses to produce areaction product that can be discerned. For example, when the agenthorseradish peroxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which is visuallydetectable. An antibody or antigen binding fragment may also beconjugated with biotin, and detected through indirect measurement ofavidin or streptavidin binding. It should be noted that the avidinitself can be conjugated with an enzyme or a fluorescent label.

An antibody may be labeled with a magnetic agent, such as gadolinium.Antibodies can also be labeled with lanthanides (such as europium anddysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody mayalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags). Insome embodiments, labels are attached by spacer arms of various lengthsto reduce potential steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. Theradiolabel may be used for both diagnostic and therapeutic purposes. Forinstance, the radiolabel may be used to detect FGFR4 by x-ray, emissionspectra, or other diagnostic techniques. Examples of labels forpolypeptides include, but are not limited to, the followingradioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁸S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In¹²⁵I, ¹³¹I.

An antibody can also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups may be useful to improve the biologicalcharacteristics of the antibody, such as to increase serum half-life orto increase tissue binding.

Toxins can be employed with the monoclonal antibodies described hereinto produce immunotoxins. Exemplary toxins include ricin, abrin,diphtheria toxin and subunits thereof, as well as botulinum toxins Athrough F. These toxins are readily available from commercial sources(for example, Sigma Chemical Company, St. Louis, Mo.). Contemplatedtoxins also include variants of the toxins described herein (see, forexample, see, U.S. Pat. Nos. 5,079,163 and 4,689,401). In oneembodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Pat. No.5,602,095). As used herein “Pseudomonas exotoxin” refers to afull-length native (naturally occurring) PE or a PE that has beenmodified. Such modifications can include, but are not limited to,elimination of domain Ia, various amino acid deletions in domains Ib, IIand III, single amino acid substitutions and the addition of one or moresequences at the carboxyl terminus (for example, see Siegall et al., J.Biol. Chem. 264:14256-14261, 1989).

PE employed with the monoclonal antibodies described herein can includethe native sequence, cytotoxic fragments of the native sequence, andconservatively modified variants of native PE and its cytotoxicfragments. Cytotoxic fragments of PE include those which are cytotoxicwith or without subsequent proteolytic or other processing in the targetcell. Cytotoxic fragments of PE include PE40, PE38, and PE35. Foradditional description of PE and variants thereof, see for example, U.S.Pat. Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and5,854,044; PCT Publication Nos. WO 99/51643 and WO 2014/052064; Pal etal., Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J.Biol. Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta1333:C1-C6, 1997.

Also contemplated herein are protease-resistant PE variants and PEvariants with reduced immunogenicity, such as, but not limited to PE-LR,PE-6X, PE-8X, PE-LR/6X and PE-LR/8X (see, for example, Weldon et al.,Blood 113(16):3792-3800, 2009; Onda et al., Proc Natl Acad Sci USA105(32):11311-11316, 2008; and PCT Publication Nos. WO 2007/016150, WO2009/032954 and WO 2011/032022, which are herein incorporated byreference).

In some examples, the PE is a variant that is resistant to lysosomaldegradation, such as PE-LR (Weldon et al., Blood 113(16):3792-3800,2009; PCT Publication No. WO 2009/032954). In other examples, the PE isa variant designated PE-LR/6X (PCT Publication No. WO 2011/032022). Inother examples, the PE variant is PE with reducing immunogenicity. Inyet other examples, the PE is a variant designated PE-LR/8M (PCTPublication No. WO 2011/032022).

Modification of PE may occur in any previously described variant,including cytotoxic fragments of PE (for example, PE38, PE-LR andPE-LR/8M). Modified PEs may include any substitution(s), for one or moreamino acid residues within one or more T-cell epitopes and/or B cellepitopes of PE.

The antibodies described herein can also be used to target any number ofdifferent diagnostic or therapeutic compounds to cells expressing FGFR4on their surface. Thus, an antibody of the present disclosure can beattached directly or via a linker to a drug that is to be delivereddirectly to cells expressing cell-surface FGFR4. This can be done fortherapeutic, diagnostic or research purposes. Therapeutic agents includesuch compounds as nucleic acids, proteins, peptides, amino acids orderivatives, glycoproteins, radioisotopes, lipids, carbohydrates, orrecombinant viruses. Nucleic acid therapeutic and diagnostic moietiesinclude antisense nucleic acids, derivatized oligonucleotides forcovalent cross-linking with single or duplex DNA, and triplex formingoligonucleotides.

Alternatively, the molecule linked to an anti-FGFR4 antibody can be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (for example, anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art (see, for example, U.S. Pat. No. 4,957,735;Connor et al., Pharm. Ther. 28:341-365, 1985).

Antibodies described herein can also be covalently or non-covalentlylinked to a detectable label. Detectable labels suitable for such useinclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels include magnetic beads, fluorescent dyes (for example,fluorescein isothiocyanate, Texas red, rhodamine, green fluorescentprotein, and the like), radiolabels (for example, ³H, ¹²⁵I, ³⁵S, ₁₄C, or³²P), enzymes (such as horseradish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (such as polystyrene,polypropylene, latex, and the like) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

IX. Immunoliposomes

Immunoliposomes are antibody-conjugated liposomes that can be used todeliver cytotoxic agents or other anti-cancer agents directly to tumorcells via binding of the antibody to a tumor specific antigen expressedon the surface of tumor cells.

The liposomal component of an immunoliposome is typically a lipidvesicle of one or more concentric phospholipid bilayers. In some cases,the phospholipids are composed of a hydrophilic head group and twohydrophobic chains to enable encapsulation of both hydrophobic andhydrophilic drugs. Conventional liposomes are rapidly removed from thecirculation via macrophages of the reticuloendothelial system (RES). Togenerate long-circulating liposomes, the composition, size and charge ofthe liposome can be modulated. The surface of the liposome may also bemodified, such as with a glycolipid or sialic acid. For example, theinclusion of polyethylene glycol (PEG) significantly increasescirculation half-life. Liposomes for use as drug delivery agents,including for preparation of immunoliposomes, have been described in theart (see, for example, Paszko and Senge, Curr Med Chem 19(31)5239-5277,2012; Immordino et al., Int J Nanomedicine 1(3):297-315, 2006; U.S.Patent Application Publication Nos. 2011/0268655; 2010/00329981).

Antibodies or antibody fragments can be conjugated to a suitableliposome according to standard methods known in the art. For example,conjugation can be either covalent or non-covalent. In some embodiments,the antibody or antibody fragment is attached to a stericallystabilized, long circulation liposome via a PEG chain. Coupling ofantibodies or antibody fragments to a liposome can also involvethioester bonds, for example by reaction of thiols and maleimide groups.Cross-linking agents can be used to create sulfhydryl groups forattachment of antibodies or antibody fragments (Paszko and Senge, CurrMed Chem 19(31)5239-5277, 2012).

X. Compositions and Methods of Use

Compositions are provided that include one or more of the disclosedantibodies that bind (for example specifically bind) FGFR4 in a carrier.Compositions comprising ADCs, CARs (and CTLs comprising CARs),bispecific antibodies, immunoliposomes and immunoconjugates are alsoprovided. The compositions can be prepared in unit dosage forms foradministration to a subject. The amount and timing of administration areat the discretion of the treating clinician to achieve the desiredoutcome. The antibody, ADC, CAR, CTL, bispecific antibody,immunoliposome or immunoconjugate can be formulated for systemic orlocal (such as intra-tumor) administration. In one example, the antibodyis formulated for parenteral administration, such as intravenousadministration.

The compositions for administration can include a solution of theantibody, ADC, CAR, CTL, bispecific antibody, immunoliposome orimmunoconjugate in a pharmaceutically acceptable carrier, such as anaqueous carrier. A variety of aqueous carriers can be used, for example,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of antibody in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the subject's needs.

A typical pharmaceutical composition for intravenous administrationincludes about 0.1 to 10 mg of antibody (or ADC, CAR, bispecificantibody or immunoconjugate) per subject per day. Dosages from 0.1 up toabout 100 mg per subject per day may be used, particularly if the agentis administered to a secluded site and not into the circulatory or lymphsystem, such as into a body cavity or into a lumen of an organ. Actualmethods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remington's Pharmaceutical Science, 19th ed., MackPublishing Company, Easton, Pa. (1995).

Antibodies (or other therapeutic molecules) may be provided inlyophilized form and rehydrated with sterile water beforeadministration, although they are also provided in sterile solutions ofknown concentration. The antibody solution is then added to an infusionbag containing 0.9% sodium chloride, USP, and in some cases administeredat a dosage of from 0.5 to 15 mg/kg of body weight. Considerableexperience is available in the art in the administration of antibodydrugs, which have been marketed in the U.S. since the approval ofRITUXAN™ in 1997. Antibodies, ADCs, CARs, bispecific antibodies,immunoliposomes or immunoconjugates can be administered by slowinfusion, rather than in an intravenous push or bolus. In one example, ahigher loading dose is administered, with subsequent, maintenance dosesbeing administered at a lower level. For example, an initial loadingdose of 4 mg/kg may be infused over a period of some 90 minutes,followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infusedover a 30 minute period if the previous dose was well tolerated.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995). Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin or a drug, as a central core. In microspheres thetherapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992).

Polymers can be used for ion-controlled release of the antibody-basedcompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known (seeU.S. Pat. Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028;4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164;5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

A. Therapeutic Methods

The antibodies, compositions, CARs (and CTLs expressing CARs), ADCs,bispecific antibodies, immunoliposomes and immunoconjugates disclosedherein can be administered to slow or inhibit the growth of tumor cellsor inhibit the metastasis of tumor cells, such as rhabdomyosarcoma, lungcancer, liver cancer, breast cancer, pancreatic cancer and prostatecancer cells. In these applications, a therapeutically effective amountof a composition is administered to a subject in an amount sufficient toinhibit growth, replication or metastasis of cancer cells, or to inhibita sign or a symptom of the cancer. Suitable subjects may include thosediagnosed with a cancer that expresses FGFR4, such as, but not limitedto, rhabdomyosarcoma, lung cancer, liver cancer, breast cancer,pancreatic cancer and prostate cancer.

Provided herein is a method of treating a subject having aFGFR4-positive cancer by selecting a subject with a FGFR4-positivecancer and administering to the subject a therapeutically effectiveamount of an antibody, ADC, CAR (e.g. a CTL expressing a CAR),bispecific antibody, immunoconjugate, immunoliposome or compositiondisclosed herein. Also provided herein is a method of inhibiting tumorgrowth or metastasis of a FGFR4-positive cancer in a subject byselecting a subject with a FGFR4-positive cancer and administering tothe subject a therapeutically effective amount of an antibody, ADC, CAR(e.g. a CTL expressing a CAR), bispecific antibody, immunoconjugate,immunoliposome or composition disclosed herein. In some embodiments, theFGFR4-positive cancer is rhabdomyosarcoma, lung cancer, liver cancer,breast cancer, pancreatic cancer or prostate cancer.

A therapeutically effective amount of a FGFR4-specific antibody, ADC,CAR (e.g. a CTL expressing a CAR), bispecific antibody, immunoconjugate,immunoliposome or composition will depend upon the severity of thedisease and the general state of the patient's health. A therapeuticallyeffective amount of the antibody-based composition is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver.

Administration of the antibodies, ADCs, CARs, immunoconjugates,bispecific antibodies, immunoliposomes and compositions disclosed hereincan also be accompanied by administration of other anti-cancer agents ortherapeutic treatments (such as surgical resection of a tumor). Anysuitable anti-cancer agent can be administered in combination with theantibodies, compositions and immunoconjugates disclosed herein.Exemplary anti-cancer agents include, but are not limited to,chemotherapeutic agents, such as, for example, mitotic inhibitors,alkylating agents, anti-metabolites, intercalating antibiotics, growthfactor inhibitors, cell cycle inhibitors, enzymes, topoisomeraseinhibitors, anti-survival agents, biological response modifiers,anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents. Otheranti-cancer treatments include radiation therapy and other antibodiesthat specifically target cancer cells.

Non-limiting examples of alkylating agents include nitrogen mustards(such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard orchlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (suchas carmustine, lomustine, semustine, streptozocin, or dacarbazine).

Non-limiting examples of antimetabolites include folic acid analogs(such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine),and purine analogs, such as mercaptopurine or thioguanine.

Non-limiting examples of natural products include vinca alkaloids (suchas vinblastine, vincristine, or vindesine), epipodophyllotoxins (such asetoposide or teniposide), antibiotics (such as dactinomycin,daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), andenzymes (such as L-asparaginase).

Non-limiting examples of miscellaneous agents include platinumcoordination complexes (such as cis-diamine-dichloroplatinum II alsoknown as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocroticalsuppressants (such as mitotane and aminoglutethimide).

Non-limiting examples of hormones and antagonists includeadrenocorticosteroids (such as prednisone), progestins (such ashydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrolacetate), estrogens (such as diethylstilbestrol and ethinyl estradiol),antiestrogens (such as tamoxifen), and androgens (such as testeroneproprionate and fluoxymesterone). Examples of the most commonly usedchemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan,CCNU,

Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU,Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, suchas docetaxel), Velban, Vincristine, VP-16, while some more newer drugsinclude Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11),Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin),Xeloda (Capecitabine), Zevelin and calcitriol.

Non-limiting examples of immunomodulators that can be used includeAS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon(Genentech), GM-CSF (granulocyte macrophage colony stimulating factor;Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immuneglobulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.),SK&F 106528, and TNF (tumor necrosis factor; Genentech).

Another common treatment for some types of cancer is surgical treatment,for example surgical resection of the cancer or a portion of it. Anotherexample of a treatment is radiotherapy, for example administration ofradioactive material or energy (such as external beam therapy) to thetumor site to help eradicate the tumor or shrink it prior to surgicalresection.

B. Methods for Diagnosis and Detection

Methods are provided herein for detecting expression of FGFR4 in vitroor in vivo. In some cases, FGFR4 expression is detected in a biologicalsample. The sample can be any sample, including, but not limited to,tissue from biopsies, autopsies and pathology specimens. Biologicalsamples also include sections of tissues, for example, frozen sectionstaken for histological purposes. Biological samples further include bodyfluids, such as blood, serum, plasma, sputum, spinal fluid or urine. Abiological sample is typically obtained from a mammal, such as a humanor non-human primate.

In one embodiment, provided is a method of determining if a subject hasa FGFR4-positive cancer by contacting a sample from the subject with amonoclonal antibody disclosed herein; and detecting binding of theantibody to the sample. An increase in binding of the antibody to thesample as compared to binding of the antibody to a control sampleidentifies the subject as having a FGFR4-positive cancer.

In another embodiment, provided is a method of confirming a diagnosis ofa FGFR4-positive cancer in a subject by contacting a sample from asubject diagnosed with a FGFR4-positive cancer with a monoclonalantibody disclosed herein; and detecting binding of the antibody to thesample. An increase in binding of the antibody to the sample as comparedto binding of the antibody to a control sample confirms the diagnosis ofa FGFR4-positive cancer in the subject.

In some examples of the disclosed methods, the monoclonal antibody isdirectly labeled.

In some examples, the methods further include contacting a secondantibody that specifically binds the monoclonal antibody with thesample; and detecting the binding of the second antibody. An increase inbinding of the second antibody to the sample as compared to binding ofthe second antibody to a control sample detects a FGFR4-positive cancerin the subject or confirms the diagnosis of a FGFR4-positive cancer inthe subject.

In some cases, the cancer is rhabdomyosarcoma, lung cancer, livercancer, breast cancer, pancreatic cancer or prostate cancer, or anyother type of cancer that expresses FGFR4.

In some examples, the control sample is a sample from a subject withoutcancer. In particular examples, the sample is a blood or tissue sample.

In some cases, the antibody that binds (for example specifically binds)FGFR4 is directly labeled with a detectable label. In anotherembodiment, the antibody that binds (for example, specifically binds)FGFR4 (the first antibody) is unlabeled and a second antibody or othermolecule that can bind the antibody that specifically binds FGFR4 islabeled. As is well known to one of skill in the art, a second antibodyis chosen that is able to specifically bind the specific species andclass of the first antibody. For example, if the first antibody is ahuman IgG, then the secondary antibody may be an anti-human-IgG. Othermolecules that can bind to antibodies include, without limitation,Protein A and Protein G, both of which are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative embodiment, FGFR4 can be assayed in a biologicalsample by a competition immunoassay utilizing FGFR4 standards labeledwith a detectable substance and an unlabeled antibody that specificallybinds FGFR4. In this assay, the biological sample, the labeled FGFR4standards and the antibody that specifically bind FGFR4 are combined andthe amount of labeled FGFR4 standard bound to the unlabeled antibody isdetermined. The amount of FGFR4 in the biological sample is inverselyproportional to the amount of labeled FGFR4 standard bound to theantibody that specifically binds FGFR4.

The immunoassays and method disclosed herein can be used for a number ofpurposes. In one embodiment, the antibody that specifically binds FGFR4may be used to detect the production of FGFR4 in cells in cell culture.In another embodiment, the antibody can be used to detect the amount ofFGFR4 in a biological sample, such as a tissue sample, or a blood orserum sample. In some examples, the FGFR4 is cell-surface FGFR4. Inother examples, the FGFR4 is soluble FGFR4 (e.g. FGFR4 in a cell culturesupernatant or soluble FGFR4 in a body fluid sample, such as a blood orserum sample).

In one embodiment, a kit is provided for detecting FGFR4 in a biologicalsample, such as a blood sample or tissue sample. For example, to confirma cancer diagnosis in a subject, a biopsy can be performed to obtain atissue sample for histological examination. Alternatively, a bloodsample can be obtained to detect the presence of soluble FGFR4 proteinor fragment. Kits for detecting a polypeptide will typically comprise amonoclonal antibody that specifically binds FGFR4, such as any of theantibodies disclosed herein. In some embodiments, an antibody fragment,such as a scFv fragment, a VH domain, or a Fab is included in the kit.In a further embodiment, the antibody is labeled (for example, with afluorescent, radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that binds FGFR4. The instructionalmaterials may be written, in an electronic form (such as a computerdiskette or compact disk) or may be visual (such as video files). Thekits may also include additional components to facilitate the particularapplication for which the kit is designed. Thus, for example, the kitmay additionally contain means of detecting a label (such as enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a secondary antibody, orthe like). The kits may additionally include buffers and other reagentsroutinely used for the practice of a particular method. Such kits andappropriate contents are well known to those of skill in the art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting FGFR4 in a biological sample generallyincludes the steps of contacting the biological sample with an antibodywhich specifically reacts, under immunologically reactive conditions, toa FGFR4 polypeptide. The antibody is allowed to specifically bind underimmunologically reactive conditions to form an immune complex, and thepresence of the immune complex (bound antibody) is detected directly orindirectly.

Methods of determining the presence or absence of a cell surface markerare well known in the art. For example, the antibodies can be conjugatedto other compounds including, but not limited to, enzymes, magneticbeads, colloidal magnetic beads, haptens, fluorochromes, metalcompounds, radioactive compounds or drugs. The antibodies can also beutilized in immunoassays such as but not limited to radioimmunoassays(RIAs), ELISA, or immunohistochemical assays. The antibodies can also beused for fluorescence activated cell sorting (FACS). FACS employs aplurality of color channels, low angle and obtuse light-scatteringdetection channels, and impedance channels, among other moresophisticated levels of detection, to separate or sort cells (see U.S.Pat. No. 5,061,620). Any of the monoclonal antibodies that bind FGFR4,as disclosed herein, can be used in these assays. Thus, the antibodiescan be used in a conventional immunoassay, including, withoutlimitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Westernblot or immunoprecipitation.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

Examples Example 1: Development and Characterization of Anti-FGFR4Monoclonal Antibodies as Therapeutic Agents

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma ofchildhood with two major subtypes—embryonal (ERMS) and alveolar (ARMS),and current treatment modalities have yielded event free 5-year survivalin only 30% of the patients with high-risk disease. Therefore, there isa need for novel strategies to identify and validate clinically relevanttargets for the treatment of RMS. The fibroblast growth factor receptor4 (FGFR4) is a very attractive therapeutic target because: 1) the FGFR4gene is over expressed in RMS, 2) it is crucial for survival,proliferation, metastasis and drug resistance, 3) activating mutationsin the kinase domain lead to aggressive growth and poor survival inpatients with alveolar RMS and 4) genetic or pharmacologic inhibition ofFGFR4-mediated signaling inhibited tumor growth in vitro and in vivo.Recent reports have shown overexpression of FGFR4 in several other humanpediatric and adult cancers including liver, lung, pancreas, ovary,prostate and breast cancer.

Table 2 provides a summary of five rabbit or mouse monoclonal antibodiesthat specifically bind FGFR4. All five antibodies detect FGFR4 proteinby both ELISA and flow cytometry.

TABLE 2 Monoclonal antibodies against human FGFR4 mAb Host CloneImmunogen Species Isotype Assay Positive 29.2 hFGFR4-ECD Rabbit IgGELISA, flow cytometer 57.1 hFGFR4-ECD Rabbit IgG ELISA, flow cytometerBT53 hFGFR4 Mouse IgGbk ELISA, flow cytometer transfected cells 1G5hFGFR4-Fc Mouse ND ELISA, flow cytometer 3A11 hFGFR4-Fc Mouse ND ELISA,flow cytometer

The affinity of two of the FGFR4-specific mAbs was tested using surfaceplasmon resonance. The results are shown in Table 3.

TABLE 3 Affinity measurements of anti-FGFR4 mAbs Analyte K_(on) K_(off)Observed Ligand (mAb) [10⁴S⁻¹M⁻¹] [10⁴S⁻¹] K_(D) [10⁻⁹M] 29.2 FGFR4-ECD2.5 4.3 17 57.1 FGFR4-ECD 0.17 1.8 110

-   -   The mAb was immobilized in a CM5 chip and the binding of FGFR4        extracellular domain (FGFR4-ECD) was detected by measuring        surface plasmon resonance in a BIACORE™ instrument

The specificity of anti-FGFR4 monoclonal antibodies 29.2, BT53 and 3A11for binding cell surface FGFR4 was evaluated by flow cytometry. Themurine RMS772 cell line was transfected with a control plasmid(RMS772-puro) or a plasmid encoding full-length human wild type FGFR4(RMS772-FGFR4). Both plasmids contained a puromycin resistance gene.Cells grown in puromycin selection medium were stained with 1 μg/mLanti-hFGFR4 mAbs from rabbit (29.2) or mouse (BT53, 3A11), andsubsequently stained with fluorochrome-conjugated secondary (anti-mouseor anti-rabbit as appropriate) antibody. Flow cytometry was performedusing FACSCalibur. As shown in FIG. 1, all the three mAbs exhibitedsignificant binding to the FGFR4 transfected cells, but not to thevector control cells.

A similar experiment was carried out to evaluate binding ofFGFR4-specific mAbs to two well-characterized RMS cell lines, each cellline representing one of the two subtypes of the disease. Binding of29.2, BT53 and 3A11 to RD cells (representing ERMS) and RH30 cells(representing ARMS) was evaluated by flow cytometry. All threeantibodies bound to both cell types.

Eight ARMS cell lines and six ERMS cell lines were tested for cellsurface expression of FGFR4. The values listed in Table 4 represent meanfluorescence intensity (MFI) obtained with individual mAbs minus the MFIobtained with corresponding isotype controls for each cell line. ThemAbs were used at different concentrations and the values obtained withthe optimal concentration (1-2 μg/mL).

TABLE 4 Comparison of FGFR4 expression between ERMS and ARMS cell linesCell Line Fusion Gene Subtype 29.2* BT53* 3A11* Birch Negative Embryonal15.0 5.3 3.6 CT10 Negative Embryonal 6.6 13.9 10.1 RD Negative Embryonal44.8 12.1 4.0 Rh36 Negative Embryonal 20.6 0.2 0.2 TTC442 NegativeEmbryonal 12.9 0.6 0.1 JR.seq PAX3/FOX01 Embryonal 20.2 51.2 36.3 RJ3.UKPAX3/FOX01 Alveolar 22.3 16.9 11.9 Rh30 PAX3/FOX01 Alveolar 69.2 34.717.6 Rh41 PAX3/FOX01 Alveolar 22.9 8.2 5.3 Rh41.UK PAX3/FOX01 Alveolar15.6 14.0 9.2 Rh5.UK PAX3/FOX01 Alveolar 13.0 13.2 8.9 Rh5 PAX3/FOX01Alveolar 17.0 3.2 2.1 SCMC.UK PAX3/FOX01 Alveolar 12.9 10.2 7.1T91-95.UK PAX7/FOX01 Alveolar 58.1 0.2 0.1 *ΔMFI with indicated mAb

FGFR4 expression was also evaluated in fresh and short-term culturedARMS tumor cells. Fresh tumor cells isolated from a metastatic breastnodule of a patient with ARMS were incubated with rabbit IgG (as acontrol) or the FGFR4-specific mAb 29.2, and subsequently stained withfluorochrome-conjugated goat anti-rabbit IgG. The fresh tumor cellsexhibited low levels of FGFR4 expression. The tumor cells were alsocultured for about one month in the presence of irradiated fibroblasts(3T3-J2) and ROCK inhibitor Y-27632. The cultured cells were harvestedand stained for FGFR4 as described above. The short-term cultured ARMStumor cells exhibited higher levels of FGFR4 expression than the freshtumor cells.

In another study, binding of anti-FGFR4 monoclonal antibodies to RMScell lines RH30 and RD was evaluated. Each RMS cell line was incubatedwith increasing concentrations of mAb 29.2 (0, 0.0001, 0.001, 0.01, 0.1,1 and 10 μg/ml) or mAb BT53 (0, 0.00032, 0.0016, 0.008, 0.04, 0.2 and 1μg/ml). Significant binding was observed with as little as 10 ng/mL ofmAb. The results also demonstrated that ARMS cell lines expressed higherlevels of cell surface FGFR4 than the ERMS cell lines.

To determine whether cell-surface binding of the FGFR4-specific mAbsleads to FGFR4 internalization, the follow study was carried out. RMScell lines (RMS772-WtFGFR4, RH30, RH41, RD and CT10) were incubated withsaturating amounts of mAb 29.2 or BT53 at 40° C. After washing, thecells were kept at 40° C. or further incubated at 37° C. for 0, 15, 30,60 or 120 minutes in the presence of 10 μM phenylarsine oxide (PAO), aninhibitor of receptor-mediated endocytosis, or its diluent DMSO, ormedium only. Subsequently, all cells were stained with an appropriate(anti-rabbit or anti-mouse) fluorochrome-conjugated secondary antibody.As shown in FIG. 2, binding of 29.2 or BT53 led to FGFR4internalization.

Conclusions

The cell surface expression of FGFR4 protein in well characterized RMScell lines, as well as in freshly isolated tumor cells from a patient,indicates that FGFR4 can serve as a therapeutic target forantibody-mediated intervention of RMS. The specificity of the anti-FGFR4mAbs described herein indicates that one or more of these mAbs can bedeveloped into a therapeutic agent. The finding that cell-surface FGFR4facilitated rapid internalization of the membrane bound mAb encouragesthe development of antibody drug conjugates, immunotoxins and chimericantigen receptor bearing T cells (CAR-T cells) and their evaluation astherapeutic agents.

Example 2: FGFR4 as a Therapeutic Target for Monoclonal Antibody BasedIntervention in Rhabdomyosarcoma

This example describes rabbit, mouse and human monoclonal antibodiesthat specifically bind FGFR4 and their characterization.

Two rabbit (29.2 and 57.1), three mouse (BT53, 3A11 and 1G5) and 10human (M408, M409, M410, M412, M414, M415, M417, M418, M422 and M424)anti-FGFR4 monoclonal antibodies were identified. The immunogen used todevelop the rabbit and human antibodies was hFGFR4-ECD. Mouse mAb BT53was developed using hFGFR4-transfected cells as the antigen, while mousemAbs 3A11 and 1G5 were generated using hFGFR4-Fc as the immunogen. Themouse and rabbit antibodies were produced using hybridoma technology.Using recombinant DNA technology, a human immunoglobulin library wasselected using FGFR4 extracellular domain (FGFR4 ECD) to derive humananti-FGFR4 mAbs.

FGFR4 is overexpressed in RMS cell lines and RMS tumors in patients,compared to normal tissues. FGFR4 overexpression in RMS tumors and RMSxenograft tissue was further demonstrated by IHC on tissue microarrays.Rh30 (ARMS), Rh41 (ARMS), Rh18 (ERMS) and Rh36 (ERMS) tumor xenografttissue exhibited positive staining with anti-FGFR4 mAb 29.2. RMS tumorsfrom calf, cheek, intra-abdominal, nasopharynx, flank and nose tissuealso stained positive by IHC using mAb 29.2.

FGFR4 expression was further evaluated in total lysates of normal tissueand RMS cell lines. As shown in FIG. 3, FGFR4 expression issignificantly higher in RMS cell lines (RH30, RD, RHS, RH28 and RMS559)than in normal tissues (heart, stomach, bladder, lung, liver,cerebellum, pancreas, colon, kidney and spleen).

To evaluate cell-surface expression of FGFR4, the murine RMS772 cellline was transfected with a control plasmid (RMS772-puro) or with aplasmid expressing full-length human FGFR4 (RMS772-FGFR4). Both plasmidscontained a puromycin resistance gene. Cells grown in puromycinselection medium were stained with 1 μg/mL anti-hFGFR4 mAbs from rabbit(29.2) or mouse (BT53), and subsequently stained withfluorochrome-conjugated secondary antibody. Flow cytometry was performedusing FACSCalibur. All the three mAbs exhibited significant binding tothe FGFR4 transfected cells, but not to the vector control cells. Normalrabbit IgG and mouse IgG were used as isotype controls. In addition, RMScell lines were incubated with different amounts of anti-FGFR4 mAb 29.2(0, 0.0001, 0.001, 0.01, 1 and 10 μg/ml) or mAb BT53 (0, 0.00032,0.0016, 0.008, 0.04, 0.04 or 1 μg/ml). Significant binding was observedwith as little as 10 ng/mL of each mAb. It was further noted that ARMScell lines expressed higher levels of cell surface FGFR4 than the ERMScell lines.

As described in Example 1, binding of anti-FGFR4 mAb inducedinternalization of cell-surface FGFR4. Therefore, an experiment wascarried out to determine whether an anti-FGFR4 mAb conjugated to asecondary antibody-drug conjugate (ADC) could mediate cytotoxicity ofRMS cells. RMS cell line RH30 was incubated with murine IgG or BT53 mAbat a concentration of 0.0001, 0.001, 0.01, 0.1, 1, 10 or 100 nM.Subsequently, secondary ADC (anti-mouse-Fc-drug) was added at 6.6 nM(FIG. 4). Dose-dependent cytotoxic activity was observed following theaddition of secondary ADC. Among the two drugs tested, DMDM showed morepotent activity than MMAF.

Conclusions

IHC analysis showed that FGFR4 is highly expressed in RMS cell lines andxenograft tissue compared to normal tissues. In addition, a differentindependent and more sensitive MSD assay also provided confirmation ofhigh FGFR4 expression on RMS cell lines compared to normal tissues. Thecell-surface expression of FGFR4 protein in well-characterized RMS cellsindicates that FGFR4 can serve as a therapeutic target forantibody-mediated intervention of RMS. The specificity of the anti-FGFR4mAbs disclosed herein indicate that these mAbs can be used astherapeutic agents. The data disclosed herein also demonstrated thatcell-surface FGFR4 facilitated rapid internalization of themembrane-bound mAb and secondary ADC mediated cytotoxicity. Thus, thedisclosed anti-FGFR4 mAbs can be used to develop ADCs for the treatmentof RMS and other cancers expressing FGFR4.

Example 3: Cytotoxic Activity Mediated by FGFR4-Specific CARs

This example describes two FGFR4-specific CARs that include the scFvsequence of the 29.2 or 57.1 antibody and their cytotoxicity againstFGFR4-positive cells.

The 29.2 L and 57.1 L CARs include an N-terminal signal peptide, the29.2 scFv or 57.1 scFv sequences (respectively) and a CH2CH3 spacer. Asshown in FIG. 5, transduced T cells are capable of expressing the FGFR4CARs.

The FGFR4 CARs were evaluated in ⁵¹Cr release assays. The cytotoxicityof T cells expressing the FGFR4 CARs 29.2 L and 57.1 L (at 10 dayspost-activation) was evaluated. Percent lysis of rhabdomyosarcoma cells(RH41), osteosarcoma cells (143B) and myelogenous leukemia cells (K562)is shown in FIG. 6. Both FGFR4 CARs are cytotoxic against FGFR4-positivecells.

Example 4: Cytotoxic Activity Mediated by FGFR4-Specific Secondary ADCs

This example describes secondary ADCs comprising the anti-FGFR4monoclonal antibodies BT53 and 3A11 and their cytotoxicity againstFGFR4-expressing cells.

Secondary ADCs comprising BT53 or 3A11 bound to a secondary antibodyconjugated to a drug (either MMAF or DMDM) were tested. As shown in FIG.7, both secondary ADCs were cytotoxic to the FGFR4-positive RH30 cellline. FIG. 8 shows that the BT53 secondary ADCs were also cytotoxic toRMS-559 cells.

Next, the specificity of the FGFR4 ADCs was evaluated. The BT53 and 3A11secondary ADCs comprising the drug DMDM were tested with FGFR4-positiverhabdomyosarcoma cells (RH30) and FGFR4-negative human skeletal musclecells (SKMC) cells. The results are shown in FIG. 9. The FGFR4 secondaryADCs were cytotoxic only to the FGFR4 expressing cells. This finding wasconfirmed by evaluating the growth of FGFR4-positive RH30 cells andFGFR4-negative SKMC cells in the presence of the BT53 monoclonalantibody or the BT53 secondary ADC. As shown in FIG. 10A, the BT53secondary ADC inhibited growth of the RH30 cells, while antibody alonedid not. Neither the BT53 monoclonal antibody nor the BT53 secondary ADCinhibited growth of the FGFR4-negative SKMC cells (FIG. 10B).

Example 5: Cytotoxic Activity Mediated by FGFR4-Specific M410 and M412CARs

This example describes three FGFR4-specific CARs that include the scFvsequences of either M410 or M412.

Three CARs were generated and tested: M410 long, M412 short and M412long. Each of the FGFR4-specific CARs described in this example includea CD8 transmembrane domain, CD137 (4-1BB) signaling domain, and a CD3zeta signaling domain (collectively having the amino acid sequence ofSEQ ID NO: 66). The M410 long and M412 long CARs also include a linkerdomain composed of CH2CH3 (SEQ ID NO: 36).

T cells expressing the CARs were evaluated for their ability to inducecytotoxicity and IFN-γ release of FGFR4-expressing cells. Target RH30(FGRR4+/CD22-) and Raji (FGFR4-/CD22+) cells were transduced withluciferase and the CELLTITER-GLO™ assay was used to measure the numberof viable cells. A CD22-specific CAR was used as a control. Percentspecific lysis induced by each CAR is shown in FIGS. 11A and 11B.FGFR4-specific CARs induced lysis of FGFR4-postive RH30 cells, but notFGFR4-negative Raji cells. The CD22-specific CAR was capable of inducingsignificant cell lysis of CD22+Raji cells, but induced only low levelsof lysis when tested with CD22-negative RH30 cells.

IFN-γ release induced by the FGFR4-specific CARs was also tested inRH30, Raji, SKES1 and K562 cells. SKES1 cells are a Ewing's sarcomacells line with detectable (by Western blot) levels of FGFR4, but FGFR4expression in these cells is not as high as in RH30 cells. K562 cellsare FGFR4-negative. As shown in FIG. 12, M410 long induced the greatestlevel of IFN-γ release in FGFR4-positive cells. Background IFN-γ releasewas also observed on K562 cells. The CD22-specific CAR induced IFN-γrelease of only CD22-positive Raji cells.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A chimeric antigen receptor (CAR) comprising: a monoclonal antibodythat binds fibroblast growth factor receptor 4 (FGFR4), orantigen-binding fragment thereof, comprising a variable heavy (VH)domain and a variable light (VL) domain, wherein the VH domain of theantibody comprises the CDR sequences of SEQ ID NO: 3 and the VL domainof the antibody comprises the CDR sequences of SEQ ID NO: 4; atransmembrane domain; and a signaling domain.
 2. The CAR of claim 1,wherein the CDR sequences are determined using the IMGT, Kabat orChothia numbering scheme.
 3. The CAR of claim 2, wherein the VH domainof the antibody comprises residues 26-33, 52-59 and 98-111 of SEQ ID NO:3 and the VL domain of the antibody comprises residues 27-32, 50-52 and88-98 of SEQ ID NO:
 4. 4. The CAR of claim 1, wherein the amino acidsequence of the VH domain is at least 90% identical to SEQ ID NO: 3 andthe amino acid sequence of the VL domain is at least 90% identical toSEQ ID NO:
 4. 5. The CAR of claim 1, wherein the amino acid sequence ofthe VH domain comprises SEQ ID NO: 3 and the amino acid sequence of theVL domain comprises SEQ ID NO:
 4. 6. The CAR of claim 1, wherein theantigen-binding fragment is a single chain variable fragment (scFv). 7.The CAR of claim 1, wherein the monoclonal antibody or antigen-bindingfragment is a fully human antibody or antigen-binding fragment.
 9. TheCAR of claim 1, wherein the monoclonal antibody or antigen-bindingfragment is a chimeric, synthetic, or humanized antibody orantigen-binding fragment.
 10. The CAR of claim 1, wherein thetransmembrane domain comprises a CD28 or a CD8 transmembrane domain. 11.The CAR of claim 10, wherein: the CD28 transmembrane domain comprisesthe amino acid sequence of SEQ ID NO: 57; or the CD8 transmembranedomain comprises the amino acid sequence of SEQ ID NO: 60 or SEQ ID NO:61.
 12. The CAR of claim 1, wherein the signaling domain comprises aCD28, CD137 or CD3ξ signaling domain.
 13. The CAR of claim 12, wherein:the CD28 signaling domain comprises the amino acid sequence of SEQ IDNO: 58; the CD137 signaling domain comprises the amino acid sequence ofSEQ ID NO: 62 or SEQ ID NO: 63; or the CD3ξ signaling domain comprisesthe amino acid sequence of SEQ ID NO:
 64. 14. The CAR of claim 1,wherein the transmembrane and signaling domains comprise the amino acidsequence of SEQ ID NO: 59, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO:67.
 15. An isolated cell expressing the CAR of claim
 1. 16. The isolatedcell of claim 15, which is a cytotoxic T lymphocyte (CTL).
 17. Acomposition comprising the CAR of claim 1 and a pharmaceuticallyacceptable carrier.
 18. A method of inhibiting tumor growth ormetastasis of a FGFR4-positive cancer, comprising selecting a subjectwith a FGFR4-positive cancer and administering to the subject atherapeutically effective amount of the CAR of claim
 1. 19. A method oftreating a FGFR4-positive cancer in a subject, comprising selecting asubject with a FGFR4-positive cancer and administering to the subject atherapeutically effective amount of the CAR of claim
 1. 20. The methodof claim 19, wherein the FGFR4-positive cancer is a rhabdomyosarcoma(RMS), lung cancer, liver cancer, breast cancer, pancreatic cancer orprostate cancer.